System, method, and apparatus facilitating automated modular manufacture of cell therapy

ABSTRACT

A rigid cartridge for housing and removably coupling with a soft body bioreactor is provided. The rigid cartridge includes rigid walls defining a rigid housing. The rigid walls expose a fixed interior configured to house the soft body. An interior surface of a first rigid wall includes a first mating mechanism to engage a second mating mechanism of the soft body to removably coupled with the rigid cartridge and accommodate the soft body in the fixed interior of the rigid cartridge. A rigid wall includes a planar upper surface including an aperture. Each aperture is encompassed by an annular hood protruding upwardly from the substantially planar upper surface and integrally formed with the at least one rigid wall. The aperture is configured to receive and fixed engage a corresponding connector. Each connector includes a rigid coupling mechanism that provides communication with a corresponding plurality of ports of the soft body.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 63/011,927, entitled “Modular Robotic System and ModularClosed-System Architecture for the Parallel, Automatic Manufacturing ofCell Therapies,” filed Apr. 17, 2020, which is hereby incorporated byreference in its entirety for all purposes.

TECHNICAL FIELD

The present disclosure generally relates to systems and methods forfacilitating modular and parallelized manufacturing at a biologicalfoundry. More particularly, the present disclosure relates to systemsand methods designed to facilitate implementing one or more workflows ata biological foundry.

BACKGROUND

Cell therapies are next-generation drugs where live cells are used totreat a subject. This is in contrast with traditional small-molecule andbiologic drugs, where small or large molecules—but not whole livingcells—are used to treat patients. Many of the most recent and promisinginnovations in medicine are represented by cell therapies in which thecells of a subject (either the patient or a donor) are extracted,genetically engineered in a lab, grown in an incubator, and finallyinfused in the patient in order to achieve a therapeutic effect.However, despite the life-saving effects of many cell therapies, thereare significant bottlenecks to their widespread adoption. For instance,one obstacle is represented by the current limits in manufacturingcapacity for cell therapies. Conventional cell therapy productionprocesses are still largely labor-based and inefficient.

Traditionally, cell therapies are produced with labor-intensiveprocesses. These conventional processes require not only a large numberof manufacturing operators, but also the employment of highly skilled(and expensive) technicians. These constraints make it particularlydifficult to manufacture cell therapies at an industrial scale. Celltherapy manufacturing processes are low-scale and labor-intensivebecause they were originally developed in the context of academicresearch. The original lab processes—which were developed to demonstratethe feasibility of cell therapies—were then hastily modified andretrofitted in order to fulfill regulatory requirements and achieve goodmanufacturing practices.

This conventional approach allowed drug manufacturers to bring to themarket the first approved cell therapies. However, this labor-intensive,lab-oriented approach is unsuitable to achieve industrial scale. Attheir core, current cell manufacturing processes were designed to bemanually completed by highly trained personnel—such as the researchersthat conduct scientific experiments in an academic environment.Requiring this type of skillset becomes a disadvantage in an industrialsetting. Cell manufacturing processes depend on highly trained, highlyeducated manual labor, and this makes them incompatible with theefficiency of mass-manufacturing industrial processes.

The dominant conventional approach to cell manufacturing is based on aset of separate individual pieces of manufacturing equipment placed on aclean room bench. This manufacturing process still looks exactly like aresearch laboratory, where all the machinery is manually operated anddirectly supervised by highly skilled operators. In order to execute thecell manufacturing processes, these skilled operators gown up, enter aclean room, and manually activate the machines. The operators alsotransfer the batch material from machine to machine, manually sample thebatches to perform quality control testing, ensure that reagents aredelivered to the cells, and ensure that waste material is removed. Thislabor-based conventional approach is very different from theorganization of industrial-scale processes, where most tasks areautonomously executed by specialized machinery, which is supervised byordinary manufacturing technicians (not engineers, nor scientists).

As such, the conventional labor-based approach to cell therapymanufacturing has at least three fundamental limits. First, theconventional approach is not scalable and not robust to operatorvariability. Because the conventional approach is extremelylabor-intensive, cell therapy manufacturing is limited to small-scaleapplications. Increasing throughput beyond a few hundred products peryear has proven extremely difficult, because such an effort wouldrequire hiring, training, retaining, and managing a large number ofhighly skilled, expensive operators. Moreover, labor-based processes aretypically unable to reach industrial scale, and cell manufacturing isnot an exception. This pronounced reliance of labor presents additionaldisadvantages, including the fact that—because of operatorvariability—the yield and the features of the finished cell therapyproduct are hard to predict and to control. This operator variabilitymakes scaling the process of manufacturing cell therapy products evenharder—particularly in terms of margins, in which a higher number ofrejected batches increases the cost per batch.

Additionally, the conventional approach to manufacturing cell therapyproducts is inefficient. Since individual machines for the cell therapymanufacturing process are utilized in series (e.g., the machines areused one at a time, with a single batch manually moved from a piece ofmachinery to the next), when a machine is active all the others areidle. This results in a low utilization rate for all machines, sincemost of the machines are waiting for the batch to arrive, while a singlemachine is being used. The problem of a very low utilization rate isparticularly evident for cell manufacturing processes, which arecharacterized by machines with markedly different cycle times. Morespecifically, systems like bioreactors process a single batch for weeks,while machines like thawing and freezing systems are only used for a fewhours on a single batch. This results in utilization rates that are evenlowed for the faster machines—because the slower machines are thebottleneck and limit the rate of the rest of the serial process.

Finally, the conventional approach to manufacturing cell therapyproducts has low throughput. Because the process is managed and executedby human operators, only one batch can be produced at any given time ona serial production line. For instance, if two batches were manufacturedat the same time on the same production line, in fact, there would behigh risk of cross-contamination or of mix-up errors by the operators.Since all the serial machines are used for just one product at a time,the resulting throughput of the production line is extremely low. As areference, typically a cell therapy product takes two to three weeks tobe manufactured. This means that, in order to avoid mix-ups, a wholeproduction line must be reserved for a single product for about half ofa month—a rate that is incompatible with industrial scale. Because ofthis temporal constraint, a whole manufacturing suite (typicallyconsisting of about 1,000 square feet of clean room space) must bereserved for a single serial production line. Therefore, the only way toincrease throughput via this conventional approach is by creatingfacilities with multiple independent suites that replicate the sameprocess. However, each suite can only handle one product at a time,occupies significant clean room space, and is entirely operated byskilled labor. As such, this conventional approach is not scalable, andnot suitable to manufacture more than a few hundreds of cell therapiesper year—with very high production costs.

One solution to this conventional approach are closed system celltherapy machines that have been developed to attempt to address theshortcomings of the traditional approach. However, even this solution isstill labor-intensive and inadequate to reach industrial scale. Forinstance, this solution can be described as an end-to-end serial systemthat is contained into a single machine. Different parts of the samemachine perform the different steps of the production process. In otherwords, a single piece of equipment contains all the sub-systems that areneeded to perform the cell manufacturing process. An intricate set oftubes connects all of these systems, so that the cell therapy product(which is typically in liquid form) can be transferred from onesub-system to the next without being exposed to the externalenvironment, which provides the closed system.

However, these end-to-end, closed systems are sold as a unique piece ofmachinery. As such, the machinery cannot be modified by the buyer: oncea system is bought, the buyer is constrained to run the exact processfor which that machine was designed. Additionally, the machinery stillneeds to be operated by a highly skilled technician, who needs toperform a complicated set of actions to set up, monitor, and manage themanufacturing process. More specifically, highly trained operators setup the intricate network of tubes that is required by each batch. Theseoperators are also tasked with opening and closing the valves thatregulate the flow of material from one part of the system to the next.Furthermore, technicians also manually sample the batch, whenevertesting is needed for quality control.

As such, this prior closed system solution suffers disadvantages, inthat the closed system solution is overcomplicated. Setting up dozens oftubes, liquid reservoir bags, and reagents requires highly trainedlabor. This setting up process also takes a long time—even for a skilledtechnician—to set up, operate, and supervise the machinery. This resultsin the need for a number of operators that increases proportionally tothe number of production system—making it impossible to achieveindustrial scale and contain manufacturing costs.

Furthermore, the prior closed system solution is inefficient. Since thearchitecture of the closed system is still serial, this approach suffersof the same efficiency constraints as the dominant (bench-based)approach. At any given time, most of the subsystems inside of theend-to-end machine are unused. This happens because only one system canbe used at a time—this is a serial production line with the hard limitof a single product per production run. Moreover, since some parts ofthe process are particularly slow (for example, the expansion of thecells into a bioreactor), the “aster subsystems are characterized by aneven lower utilization rate than the slower subsystems of the machinery.

Additionally, this closed system lacks design flexibility. Thisinflexibility drawback is typical of closed systems that are builtspecifically to execute a particular process. Once the machinery isbought, it is not possible to replace an outdated subsystem with abetter one (for example, a subsystem that performs a task better, orwith a higher throughput). Any modification to the original closedsystem machinery requires massive engineering and retooling costs,comparable to building a whole new end-to-end system from scratch. Thislack of flexibility is particularly disadvantageous in the case of celltherapy manufacturing—where processes are often tuned and improvement atall stages of clinical development.

Moreover, since each closed system is end-to-end and can onlymanufacture a single product at a time, the only way to increasethroughput is to buy more of these closed systems. This in turn worsensthe above-mentioned complexity and underutilization problems. In otherwords, deploying more complex systems increases the need for skilledoperators, which in turn increases the cost of manufacturing. Since eachmachine is largely underutilized (only one subsystem is active at anygiven time), chronic underutilization also characterizes a facility thatis equipped with multiple end-to-end systems. Furthermore, conventionaldocking station designs does lend themselves to application in celltherapy manufacturing. For instance, conventional docking stations donot include passive compliance and passive damping systems. Instead,conventional docking stations utilize rigid features, jigs, pins,chamfers, and the like.

Prior solutions, like wedges and chamfers, are easy for robotic systemsto interface with. However, wedges and chamfers can only keep a part inplace due to gravity. This is inadequate when there are vibrations(i.e., the wedge could move the part outside of the docking station), orwhen forces perpendicular to gravity could be exerted on the part. Forexample, if the part is pushed from the side, it can easily slide out ofa chamfered docking station. On the other hand, prior solutions likelocating pins are hard to operate for robots, because: the locating pinsrequire high accuracy; and the locating pins have a high rigidity, whichmeans that they are not tolerant to misalignments. This affectsnegatively the repeatability of the process, which would present ahigher risk of failure for pick and/or place operations conductingduring the manufacture of cell therapies.

Additionally, a major problem of labor-based cell manufacturingprocesses is that human operators need to sample each batch manually. Incell manufacturing processes, sterility must be always ensured. This isparticularly important, because cell therapies cannot be sterilized atthe end of the manufacturing process (that would kill the cells). At thesame time, guaranteeing the quality of cell manufacturing processesrequires a large number of quality control steps. And, in order toperform quality control tests, the cell therapy products must befrequently sampled (i.e. a part of the product must be removed from thebatch, while ensuring the sterility of both the sample and the product).In conventional cell manufacturing processes, sampling tasks areexecuted by human operators.

One disadvantage of this conventional approach to sampling is that humanoperators are a significant potential source of contamination for celltherapy products. Every time a batch is sampled manually, there is ahigh risk of contamination because the operator must manually remove apart of the liquid containing the cell product. Even semi-automatedsampling procedures, where an operator activates a system that performsthe sampling task, present significant risk of contamination due torequiring the presence of a human technicians in close proximity to theprocess.

Another critical issue is that sampling procedures are performedextremely frequently in cell manufacturing processes. Cell therapyproducts are sometimes sampled multiple times during a single day. Sincecell manufacturing processes have a long completion time (most requiremore than a week, and many can take up to fifteen to twenty days),manual sampling is repeated dozens of times for every single batch.Repeating risky sampling procedures with this extreme frequency greatlyincreases the risk of contamination.

Given the above background, there is a need in the art for improvedsystems, methods, and apparatuses for facilitating an improvedmanufacture of cell therapies that addresses these dilemmas.

The information disclosed in this background section is only forenhancement of understanding of the general background of the inventionand should not be taken as an acknowledgement or any form of suggestionthat this information forms the prior art already known to a personskilled in the art.

SUMMARY

Advantageously, the systems and methods detailed in the presentdisclosure address the shortcomings in the prior art detailed above.

Systems, methods, and apparatuses for broadly implementing a manufactureof a cellular-cellular engineering target at a biological foundry systemare provided.

Specifically, exemplary systems, methods, and apparatuses of the presentdisclosure directly apply to both manufacturing systems for producingsmall-cellular engineering targets, high-mix cellular engineeringtargets, personalized cellular engineering targets, or just-in-timeproduction of such targets.

One aspect of the present disclosure is directed to providing a modularbiological foundry system for the manufacture of cellular engineeringtargets, such as cell therapy products. The modular biological foundrysystem is a fully autonomous manufacturing system that includes multiplemodules accommodated by a frame. Each module performs a task in themanufacturing process of the cellular engineering targets. The cellularengineering targets are moved from a first module to a second module bya robotic material transfer system until the manufacture of the cellularengineering target is deemed completed. In some embodiments, the roboticmaterial transfer system includes an articulated handling robot and,optionally, a transport path coupled to the articled handling robot,which is surrounded by the frame. The modules are kept in place by theframe, which also creates a clean-room, sterile environment inside themodular biological foundry system. This modular architecture provided bythe frame and the modules allows for efficiently manufacturingindividualized cellular engineering targets in a sterile environment.Moreover, the modules are also fault-tolerant and flexible in design, inthat modules can be swapped as the manufacture process improves, withoutaffecting the rest of the modular biological foundry system. Inside themodular biological foundry system, the cellular engineering targets arealways accommodated inside of modular, sterile closed systems, as knownas a rigid cartridge. These cartridges enable the robotic materialtransfer system to autonomously operate the instruments that perform themanufacturing process.

Another aspect of the present disclosure is directed to providing amodular clean room biological foundry system for producing one or morecellular engineering targets. The modular clean room biological foundrysystem includes a controller and a communications interface inelectrical communication the controller and a plurality of peripheraldevices. The plurality of peripheral devices includes an articulatedhandling robot and a power supply. The articulated handling robot isconfigured to move a cell therapy cartridge between at least a firstbiological foundry instrument and a second biological foundry instrumentin a plurality of biological foundry instruments configured to produce aportion of the one or more cellular engineering targets. The modularclean room system includes a frame surrounding the articulated handlingrobot. The frame includes at least two modules in a plurality ofmodules. A first module in the at least two modules is configured toaccommodate a respective biological foundry instrument in the pluralityof biological foundry instructions. Moreover, each module in theplurality of modules includes a plurality of elongated members. Eachmodule in the plurality of modules further includes a first plurality ofcoupling mechanisms for coupling at least two elongated members in theplurality of elongated members. Additionally, each module in theplurality of modules includes a second plurality of coupling mechanismsfor removably coupling a respective elongated member in the plurality ofelongated members with the frame. Furthermore, each module in theplurality of modules includes a plurality of walls engaged with andsupported by the at least two elongated members in the plurality ofelongated members. Accordingly, the plurality of walls forms an internalvolume of a respective module sealed from an environment, such that amodular clean room biological foundry system is provided.

In some embodiments, the modular clean room biological foundry systemfurther includes a transport path coupled to the articulated handlingrobot and in electrical communication with the communications interface.The transport path extends from a first end portion of a first wall inthe plurality of walls to a second end portion of a second wall in theplurality of walls.

In some embodiments, the transport path provides one degree of freedomof movement to articulated handling robot.

In some embodiments, a first wall in the plurality of walls isconfigured as an air inflow port and a second wall in the plurality ofwalls is configured as an air outflow port.

In some embodiments, the first wall includes an air filter mechanism.

In some embodiments, the internal sealed volume is in a range from about0.3 cubic meters (m³) to about 1.5 m³.

In some embodiments, a second module in the at least two modules isdisposed above the first module in the at least two modules.

In some embodiments, the first plurality of coupling mechanism includesa gasket.

Yet another aspect of the present disclosure is directed to providing adocking device for receiving a portable cell therapy cartridge. Thedocking device includes a base including a substantially planar uppersurface configured to support a lower surface of a portable cell therapycartridge. The docking device further includes an array of a pluralityof engagement members protruding upwardly from the substantially planarupper surface of the base. Each engagement member in the array of theplurality of engagement members includes a fixed body protrudingupwardly from the substantially planar upper surface of the base.Moreover, each engagement member in the array of the plurality ofengagement members includes a spring integrally formed with the fixedbody and extending inwardly from an upper end portion of the fixed bodyoblique to both the substantially planar upper surface of the base andthe fixed body. From this, a deformable gap is formed interposingbetween a surface of the fixed body and a lower end surface of thespring. Accordingly, an upper end surface of the spring is configured toengage a surface of the portable cell therapy cartridge.

In some embodiments, the array of the plurality of engagement membersincludes at least three engagement members.

In some embodiments, the array of the plurality of engagement membersincludes twelve engagement members.

In some embodiments, each respective engagement member in the array ofthe plurality of engagement members opposes a corresponding engagementmember in the plurality of engagement members.

In some embodiments, a first distance from a first end portion of afirst engagement member in the array of the plurality of engagementmembers to a second end portion of a second engagement member in thearray of the plurality of engagement members is less than a length ofthe portable device.

In some embodiments, a maximum width of the deformable gap in anunengaged state is in range of from about 0.2 mm to about 1.5 mm.

In some embodiments, a minimum width of the deformable gap in an engagedstate is in range of from about 0 mm to about 1.0 mm.

In some embodiments, the spring of each engagement member in the arrayof the plurality of engagement members is a leaf spring.

In some embodiments, an upper surface of the spring of each engagementmember in the array of the plurality of engagement members includes adownward slope towards the substantially planar surface of the base.

In some embodiments, each engagement member in the array of theplurality of engagement members includes an elastic material.

In some embodiments, each engagement member in the array of theplurality of engagement members removably couples with the base.

In some embodiments, a thickness of the spring each engagement member inthe array of the plurality of engagement members is in a range of from 2mm to 15 mm.

In some embodiments, the thickness of the spring is constant along alength of the engagement member in the array of the plurality ofengagement members.

In some embodiments, each engagement member in the array of theplurality of engagement members includes polylactic acid (PLA),acrylonitrile butadiene styrene (ABS), polyethylene terephthalate (PET),PET glycol-modified (PETG), polyethylene cotrimethylene terephthalate(PETT), thermoplastic elastomer (TPE), thermoplastic polyurethane (TPU),thermoplastic copolyester (TPC), nylon, polycarbonate (PC), brass,copper, bronze, aluminum, or iron.

In an exemplary embodiment, the invention is directed to providing arigid cartridge for housing and removably coupling with a soft bodybioreactor. An exemplary rigid cartridge includes a plurality of rigidwalls defining a fixed interior. The plurality of rigid walls includes aplurality of rigid side walls. At least two adjacent edges in aplurality of edges formed by the plurality rigid side walls includes aradius of curvature greater than zero. A substantially planar upperrigid wall connected to an upper edge portion of each rigid side wall inthe plurality of rigid side walls. The substantially planar upper rigidwall includes a plurality of apertures. Each respective aperture in theplurality of apertures is configured to receive and fixedly engage acorresponding connector, which, in an exemplary embodiment, is a memberof a group consisting of at least three connectors. Moreover, eachrespective connector in the at least three connectors in such anembodiment provides communication with a corresponding port in aplurality of ports of the soft body bioreactor. In some embodiment, eachrespective aperture in a first subset of apertures in the plurality ofapertures is encompassed by a corresponding annular hood protrudingupwardly from the substantially planar upper rigid wall by a firstheight greater than or equal to a second height of the correspondingconnector of the at least three connectors and integrally formed withthe substantially planar upper rigid wall. Additionally, the pluralityof apertures includes the first subset of apertures. Each respectiveaperture in the first subset of apertures is configured to receive andfixedly engage a fluidic connector of the at least three connectorsconfigured to provide fluidic communication with at least a first portin the plurality of ports of the soft body bioreactor, and a secondsubset of apertures. Furthermore, in various embodiments, eachrespective aperture in the second subset of apertures is configured toreceive and fixedly engage an electrical connector of the at least threeconnectors configured to provide electrical communication with at leasta second port in the plurality of ports of the soft body bioreactor. Anexemplary rigid cartridge further includes a seamless lip defined by alower edge portion of each rigid side wall in the plurality of rigidside walls. Each interior surface in a pair of opposing interiorsurfaces of two rigid side walls in the plurality of rigid side wallsincludes a respective first mating mechanism configured to engage acorresponding second mating mechanism of the soft body bioreactor, suchthat a pair of opposing end portions of the soft body bioreactor isremovably coupled with the rigid cartridge.

In some embodiments, the electrical connector includes a pressurecontrol mechanism, a pH sensor, a dissolved oxygen sensor, a temperaturesensor, a flow rate sensor, a mass sensor, or a combination thereof.

In some embodiments, the fluidic connector includes a pressure controlmechanism, an inlet port, a sampling port, an outlet port, or acombination thereof.

In some embodiments, the fluidic connector includes a valve configuredto control a flow of a fluid through a corresponding fluidic port.

In some embodiments, a first internal diameter at an upper end portionof a respective connector is less than a second internal diameter at alower end portion of the respective connector.

In some embodiments, at least one rigid wall in the plurality of rigidwalls further includes a gate mechanism configured to provide access tothe fixed internal cavity of the rigid housing.

In some embodiments, a length of the gate mechanism is greater than orequal to a cross-sectional area of the soft body bioreactor.

In some embodiments, the gate mechanism is a hinge gate mechanism or ahinge gate mechanism.

In some embodiments, each rigid coupling mechanism protrudes from thesubstantially upper planar surface of the at least one rigid wall of therigid housing.

In some embodiments, the rigid cartridge further includes acorresponding cap for each respective connector in the plurality ofconnectors. The corresponding cap encompasses the respective connector.

In some embodiments, an exterior surface of the corresponding capincludes a third mating mechanism confirmed to engage a fourth matingmechanism of an articulated handling robot.

In some embodiments, the corresponding cap is configured to disengagethe respective connector in the plurality of connectors when subject toa lateral force.

In some embodiments, each rigid coupling mechanism of each respectiveport in the at least one electrical port is a push coupling mechanism.

In various embodiments of the present invention there is provided arigid cartridge for housing and removably coupling with a soft bodybioreactor. An exemplary rigid cartridge includes a plurality of rigidwalls defining a rigid housing including an open face forming a seamlesslip at a lower end portion of the rigid cartridge. The plurality ofrigid walls exposes a fixed interior configured to house the soft bodybioreactor. An interior surface of a first rigid wall in the pluralityof rigid walls includes a first mating mechanism configured to engage asecond mating mechanism of the soft body bioreactor. From this, the softbody bioreactor is removably coupled with the rigid cartridge andaccommodates the soft body bioreactor in the fixed interior of the rigidcartridge. Additionally, at least one rigid wall in the plurality ofrigid walls includes a substantially planar upper surface comprising atleast aperture. Each respective aperture of the at least one aperture isencompassed by an annular hood protruding upwardly from thesubstantially planar upper surface and integrally formed with the atleast one rigid wall. An exemplary at least one aperture is configuredto receive and fixed engage a corresponding connector which is a memberof a group consisting of at least one connector. Furthermore, eachrespective connector in the at least one connector includes at least onerigid coupling mechanism that provides communication with acorresponding plurality of ports of the soft body bioreactor.Additionally, the corresponding plurality of ports includes at least oneelectrical port and at least one fluidic port.

The methods and apparatuses of the present disclosure have otherfeatures and advantages which will be apparent from or are set forth inmore detail in the accompanying drawings, which are incorporated herein,and the following Detailed Description, which together serve to explaincertain principles of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary biological foundry workflow systemtopology including a computer system and a plurality of instrumentsassociated with a biological foundry, in accordance with an embodimentof the present disclosure;

FIG. 2 illustrates various modules and/or components of a computersystem, in accordance with an exemplary embodiment of the presentdisclosure;

FIG. 3 is a perspective view of a modular clean room biological foundryincluding a plurality of instruments, in accordance with an embodimentof the present disclosure;

FIG. 4 is a side view of a modular clean room biological foundry, inaccordance with an embodiment of the present disclosure;

FIG. 5 is a front view of a modular clean room biological foundrysystem, in accordance with an embodiment of the present disclosure;

FIG. 6 is a perspective view of a module of a modular clean roombiological foundry system, in accordance with an embodiment of thepresent disclosure;

FIG. 7 is a perspective view of a docking device instrument, inaccordance with an embodiment of the present disclosure;

FIG. 8 is a side view of a docking device instrument, in accordance withan embodiment of the present disclosure;

FIG. 9 is a top view of a docking device instrument, in accordance withan embodiment of the present disclosure;

FIG. 10 is a side view of an engagement member of a docking deviceinstrument, in accordance with an embodiment of the present disclosure;

FIG. 11 is a perspective view of a plurality of instruments of abiological foundry including a rigid cartridge, in accordance with anembodiment of the present disclosure;

FIG. 12 is a side view of a plurality of instruments of a biologicalfoundry including a rigid cartridge, in accordance with an embodiment ofthe present disclosure;

FIG. 13 is a side view of a plurality of instruments of a biologicalfoundry including a soft body bioreactor, in accordance with anembodiment of the present disclosure;

FIG. 14 is a perspective view of a rigid cartridge, in accordance withan embodiment of the present disclosure;

FIG. 15 is another perspective view of a plurality of instruments of abiological foundry including a rigid cartridge, in accordance with anembodiment of the present disclosure;

FIG. 16 is yet another perspective view of a plurality of instruments ofa biological foundry including a rigid cartridge, in accordance with anembodiment of the present disclosure;

FIG. 17 is a side view of a rigid cartridge, in accordance with anembodiment of the present disclosure;

FIG. 18A is a side view of another rigid cartridge, in accordance withan embodiment of the present disclosure;

FIG. 18B is a side view of yet another rigid cartridge, in accordancewith an embodiment of the present disclosure;

FIG. 18C is a side view of yet another rigid cartridge, in accordancewith an embodiment of the present disclosure;

FIG. 19A is a side view of another rigid cartridge, in accordance withan embodiment of the present disclosure;

FIG. 19B is a side view of yet another rigid cartridge, in accordancewith an embodiment of the present disclosure;

FIG. 19C is a side view of yet another rigid cartridge, in accordancewith an embodiment of the present disclosure;

FIG. 20 is a side view of yet another rigid cartridge, in accordancewith an embodiment of the present disclosure;

FIG. 21 is a schematic view of yet another rigid cartridge, inaccordance with an embodiment of the present disclosure;

FIG. 22 is a schematic view of yet another rigid cartridge, inaccordance with an embodiment of the present disclosure;

FIG. 23 is a schematic view of a modular clean room biological foundry,in accordance with an embodiment of the present disclosure; and

FIG. 24 is a schematic view of a modular clean room biological foundry,in accordance with an embodiment of the present disclosure.

It should be understood that the appended drawings are not necessarilyto scale, presenting a somewhat simplified representation of variousfeatures illustrative of the basic principles of the invention. Thespecific design features of the present invention as disclosed herein,including, for example, specific dimensions, orientations, locations,and shapes will be determined in part by the particular intendedapplication and use environment.

In the figures, reference numbers refer to the same or equivalent partsof the present invention throughout the several figures of the drawing.

DETAILED DESCRIPTION

The present disclosure provides systems, methods, and apparatuses forfacilitating automated modular manufacture of cellular engineeringtargets. Exemplary systems, methods, and apparatuses for themanufacturing of cellular engineering targets of the present disclosureincludes the advantages of modularity, flexibility, and scalability.Moreover, exemplary systems, methods, and apparatuses of the presentdisclosure retain the benefits of a conventional closed-systemprocesses, such as providing a sterile clean room environment, withoutsacrificing the aforementioned advantages. Furthermore, exemplarysystems, methods, and apparatus of the present disclosure leverageadvanced robotic features and technologies that enables thetransformation of cellular engineering target manufacturing fromlabor-based and low-throughput processes to fully industrialized,high-throughput processes with high scale, efficiency and repeatability.Accordingly, an exemplary modular biological foundry system provided bythe present disclosure offers the advantages of increased throughput, inthat multiple separate cellular engineering targets can be produced atthe same time within the modular biological foundry system. This isenabled by a parallel architecture of modules of the modular biologicalfoundry system. Adding more copies of modules that facilitate slowerprocesses decreases the overall cycle time of modular biological foundrysystem. Additionally, the systems, methods, and apparatus of exemplaryembodiments provide increased efficiency by reducing bottlenecks whenmanufacturing a cellular engineering target. Exemplary modules have highutilization rates due to the parallel architecture of the modularbiological foundry system. Organizing the modules associated with fastermanufacturing processes in groups, while adding more copies of themodules associated with slower manufacturing processes, results inreduction or elimination of production bottlenecks. Furthermore,exemplary systems, methods, and apparatus of the present disclosureprovides increased safety, such as improved sterility within a modularbiological foundry system. Since the manufacture of cellular engineeringtargets can be entirely automated and contained in a clean room (e.g.,the modular biological foundry system forms an entirely self-contained,sterile clean room space), there are no human sources of contaminationaround the cellular engineering targets. Additionally, exemplarysystems, methods, and apparatus of the present disclosure allows forhigher consistency and repeatability when manufacturing cellularengineering targets since the entire cell manufacturing process isexecuted by a robotic material transfer system within the modularbiological foundry system. This fully automated approach ensures morerepeatability and traceability for the manufacturing process. Finally,the systems, methods, and apparatus of the present disclosure provideimproved flexibility in design. An exemplary modular biological foundrysystem is designed to allow the addition of new modules, or the removalof old modules. If a more advanced module becomes available, it can beadded to modular biological foundry system without influencing the restof the system. Similarly, if an old module must be removed, it ispossible to do so without affecting the rest of the system. This makesit possible to implement different processes (by choosing theappropriate initial set of modules), increase throughput (by adding moremodules to a cluster, or by building new copies of a whole modularbiological foundry system), and improve a process (by swappingpre-existing modules with improved ones). And since the flow ofmaterials within an exemplary modular biological foundry system ismanaged by the robotic material transfer system, the sequence ofoperations in the manufacturing process can simply be updated byupdating the software that controls the modular biological foundrysystem.

Reference will now be made in detail to various embodiments of thepresent disclosure, examples of which are illustrated in theaccompanying drawing and described below. While the disclosure will bedescribed in conjunction with exemplary embodiments, it will beunderstood that the present description is not intended to limit theinvention(s) to those exemplary embodiments. On the contrary, theinvention(s) is/are intended to cover not only the exemplaryembodiments, but also various alternatives, modifications, equivalentsand other embodiments, which may be included within the spirit and scopeof the present invention as defined by the appended claims.

It will also be understood that, although the terms first, second, etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. For example, a first instrument could betermed a second instrument, and, similarly, a second instrument could betermed a first instrument, without departing from the scope of thepresent disclosure. The first instrument and the second instrument areboth instruments, but they are not the same instrument.

The terminology used in the present disclosure is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the invention. As used in the description of the inventionand the appended claims, the singular forms “a,” “an,” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will also be understood that the term “and/or”as used herein refers to and encompasses any and all possiblecombinations of one or more of the associated listed items. It will befurther understood that the terms “comprises” and or “comprising,” whenused in this specification, specify the presence of stated features,integers, steps, operations, elements, and or components, but do notpreclude the presence or addition of one or more other features,integers, steps, operations, elements, components, and/or groupsthereof.

As used herein, the term “if” may be construed to mean “when” or “upon”or “in response to determining” or “in response to detecting,” dependingon the context. Similarly, the phrase “if it is determined” or “if [astated condition or event] is detected” may be construed to mean “upondetermining” or “in response to determining” or “upon detecting [thestated condition or event]” or “in response to detecting [the statedcondition or event],” depending on the context.

Furthermore, when a reference number is given an “i^(th)” denotation,the reference number refers to a generic component, set, or embodiment.For instance, an application termed “application i” refers to the i^(th)application in a plurality of applications.

The foregoing description included example systems, methods, techniques,instruction sequences, and computing machine program products thatembody illustrative implementations. For purposes of explanation,numerous specific details are set forth in order to provide anunderstanding of various implementations of the inventive subjectmatter. It will be evident, however, to those skilled in the art thatimplementations of the inventive subject matter may be practiced withoutthese specific details. In general, well-known instruction instances,protocols, structures and techniques have not been shown in detail.

The foregoing description, for purpose of explanation, has beendescribed with reference to specific implementations. However, theillustrative discussions below are not intended to be exhaustive or tolimit the implementations to the precise forms disclosed. Manymodifications and variations are possible in view of the aboveteachings. The implementations are chosen and described in order to bestexplain the principles and their practical applications, to therebyenable others skilled in the art to best utilize the implementations andvarious implementations with various modifications as are suited to theparticular use contemplated.

In the interest of clarity, not all of the routine features of theimplementations described herein are shown and described. It will beappreciated that, in the development of any such actual implementation,numerous implementation-specific decisions are made in order to achievethe designer's specific goals, such as compliance with use case- andbusiness-related constraints, and that these specific goals will varyfrom one implementation to another and from one designer to another.Moreover, it will be appreciated that such a design effort might becomplex and time-consuming, but nevertheless be a routine undertaking ofengineering for those of ordering skill in the art having the benefit ofthe present disclosure.

An aspect of the present disclosure is directed to providing systems,methods, and apparatuses for facilitating automated modular manufactureof cell therapies.

A detailed description of an exemplary system 10 for implementing theautomated modular production of cellular engineering targets (e.g., celltherapies) at a biological foundry 200 is described in conjunction withFIG. 1 and FIG. 2. As such, FIG. 1 and FIG. 2 collectively illustrate anexemplary topology of the system 10. In the topology, there is acomputer system 100 for generating a workflow that produces a pluralityof cellular engineering targets, and providing scheduling of a pluralityof instruments (e.g., first instrument 300-1, . . . , instrument 300-Rof FIG. 1; instrument 300-1 of FIG. 7, instrument 300-2 of FIG. 8,instrument 300) in correlation with a corresponding plurality ofbiological foundry operations, and oversight of the manufacture of theplurality of cellular engineering targets at the modular biologicalfoundry system.

In some embodiments, each cellular engineering target (e.g., cellularengineering target 2 of FIG. 18B), in the context of biologicalengineering at a modular biological foundry system, is one of theobjectives of a research and development project that defines thedesired biological trait to be achieved. The cellular engineering targetcan be either quantitative or qualitative. For example, in oneembodiment, a cellular engineering target(s) can be a geneticconfiguration for a biosynthetic pathway that produces more compound ofinterest than a current level. In another embodiment, the cellularengineering target(s) is a genetic configuration for a microbial hostthat has a tolerance to an inhibitor over X mg/L.

In some embodiments, each cellular engineering target includes modifiedimmune cells or precursors thereof, such as modified T cells, includinga chimeric antigen receptor (CAR). Thus, in some embodiments, the immunecell is genetically modified at a modular biological foundry system toexpress the CAR. In some embodiments, CARs include an antigen bindingdomain, a transmembrane domain, a hinge domain, and an intracellularsignaling domain.

In some embodiments, the antigen binding domain is operably linked toanother domain of the CAR, such as the transmembrane domain or theintracellular domain, for expression in the cellular engineering target.In some embodiments, a first nucleic acid sequence encoding the antigenbinding domain is operably linked to a second nucleic acid encoding atransmembrane domain, and further operably linked to a third a nucleicacid sequence encoding an intracellular domain.

The antigen binding domains described herein can be combined with any ofthe transmembrane domains, any of the intracellular domains orcytoplasmic domains, or any of the other domains that may be included ina CAR. In some embodiments, a cellular engineering target CAR of thepresent disclosure includes a spacer domain. In some embodiments, eachof the antigen binding domain, transmembrane domain, and intracellulardomain is separated by a linker.

In the present disclosure, the cellular engineering targets generallyinclude mammalian cells, and typically include human cells. In someembodiments, the cellular engineering target is derived from the blood,bone marrow, lymph, or lymphoid organs. In some embodiments, thecellular engineering targets includes cells of the immune system, suchas cells of innate or adaptive immunity, e.g., myeloid or lymphoidcells, including lymphocytes, typically T cells and/or NK cells. In someembodiments, the cellular engineering targets include stem cells, suchas multipotent and pluripotent stem cells, including induced pluripotentstem cells (iPSCs). The cellular engineering targets typically areprimary cells, such as those isolated directly from a subject and/orisolated from a subject and frozen. In some embodiments, the cellularengineering targets include one or more subsets of T cells or other celltypes, such as whole T cell populations, CD4+ cells, CD8+ cells, andsubpopulations thereof, such as those defined by function, activationstate, maturity, potential for differentiation, expansion,recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, degree ofdifferentiation, or a combination thereof. With reference to the subjectto be treated, the cellular engineering targets be allogeneic and/orautologous. In some embodiments, the modular biological foundry systemfacilitates manufacturing the cellular engineering targets by isolatingcells from the subject, preparing the cells, processing the cells,culturing the cells, engineering the cells, and re-introducing the cellsinto the same subject, before or after cryopreservation. In someembodiments, one or more of these steps is performed at modules 208 ofmodular biological foundry system 200 by utilizing rigid cartridge 1100and a robot material transfer system.

In some embodiments, among the sub-types and subpopulations of T cellsand/or of CD4+ and/or of CD8+ T cells of the cellular engineeringtargets are naive T (T.sub.N) cells, effector T cells (T.sub.EFF),memory T cells and sub-types thereof, such as stem cell memory T(T.sub.SCM), central memory T (T.sub.CM), effector memory T (T.sub.EM),or terminally differentiated effector memory T cells, tumor-infiltratinglymphocytes (TIL), immature T cells, mature T cells, helper T cells,cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturallyoccurring and adaptive regulatory T (Treg) cells, helper T cells, suchas TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cellular engineering targets are natural killer(NK) cells. In some embodiments, the cellular engineering targets aremonocytes or granulocytes, e.g., myeloid cells, macrophages,neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

Accordingly, the present disclosure provides systems and methods forproducing or generating a cellular engineering target that is a modifiedimmune cell or precursor thereof (e.g., a T cell) of the invention fortumor immunotherapy, e.g., adoptive immunotherapy. The cellularengineering targets generally are engineered by introducing one or morenucleic acids encoding a subject CAR, dominant negative receptor and/orswitch receptor, and/or bispecific antibody, and/or combinationsthereof.

In some embodiments, one or more nucleic acids encoding the subject CAR,dominant negative receptor and/or switch receptor, and/or bispecificantibody is introduced into a cell by an expression vector. Expressionvectors including a nucleic acid sequence encoding a subject CAR,dominant negative receptor and/or switch receptor, and/or bispecificantibody, and/or combinations thereof, of the present disclosure areprovided herein. Suitable expression vectors include lentivirus vectors,gamma retrovirus vectors, foamy virus vectors, adeno associated virus(AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA,including but not limited to transposon mediated vectors, such asSleeping Beauty, Piggybak, and Integrases such as Phi31. Some othersuitable expression vectors include Herpes simplex virus (HSV) andretrovirus expression vectors.

Adenovirus expression vectors are based on adenoviruses, which have alow capacity for integration into genomic DNA but a high efficiency fortransfecting host cells. Adenovirus expression vectors containadenovirus sequences sufficient to: (a) support packaging of theexpression vector and (b) to ultimately express the subject CAR,dominant negative receptor and/or switch receptor, and/or bispecificantibody, and/or combinations thereof, in the host cell. In someembodiments, the adenovirus genome is a 36 kb, linear, double strandedDNA, where a foreign DNA sequence (e.g., a nucleic acid encoding asubject CAR, dominant negative receptor and/or switch receptor, and/orbispecific antibody, and/or combinations thereof) may be inserted tosubstitute large pieces of adenoviral DNA in order to make theexpression vector of the present invention. Additional details andinformation can be found at Danthinne et al., 2002, Gene Therapy, 7(20,pg. 1707, which is hereby incorporated by reference in its entirety.

Another expression vector is based on an adeno associated virus, whichtakes advantage of the adenovirus coupled systems. This AAV expressionvector has a high frequency of integration into the host genome.Moreover, this AAV expression can infect non-dividing cells, thus makingit useful for delivery of genes into mammalian cells, for example, intissue cultures or in vivo. The AAV vector has a broad host range forinfectivity.

Retrovirus expression vectors are capable of integrating into the hostgenome, delivering a large amount of foreign genetic material, infectinga broad spectrum of species and cell types and being packaged in specialcell lines. The retrovirus vector is constructed by inserting a nucleicacid (e.g., a nucleic acid encoding a subject CAR, dominant negativereceptor and/or switch receptor, and/or bispecific antibody, and/orcombinations thereof) into the viral genome at certain locations toproduce a virus that is replication defective. Though the retrovirusvectors are able to infect a broad variety of cell types, integrationand stable expression of the subject CAR, dominant negative receptorand/or switch receptor, and/or bispecific antibody, and/or combinationsthereof, requires the division of host cells.

Lentivirus vectors are derived from lentiviruses, which are complexretroviruses that, in addition to the common retroviral genes gag, pol,and env, contain other genes with regulatory or structural function(see, e.g., U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentiviruses include the Human Immunodeficiency Viruses (HIV-1, HIV-2)and the Simian Immunodeficiency Virus (SIV). Lentivirus vectors havebeen generated by multiply attenuating the HIV virulence genes, forexample, the genes env, vif, vpr, vpu and nef are deleted making thevector biologically safe. Lentivirus vectors are capable of infectingnon-dividing cells and can be used for both in vivo and ex vivo genetransfer and expression, e.g., of a nucleic acid encoding a subject CAR,dominant negative receptor and/or switch receptor, and/or bispecificantibody, and/or combinations thereof.

Expression vectors including a nucleic acid of the present disclosurecan be introduced into a host cell by any means known to persons skilledin the art. The expression vectors may include viral sequences fortransfection, if desired. Alternatively, the expression vectors may beintroduced by fusion, electroporation, biolistic, transfection,lipofection, or the like. The host cell may be grown and expanded inculture before introduction of the expression vectors, followed by theappropriate treatment for introduction and integration of the vectors.The host cells are then expanded and may be screened by virtue of amarker present in the vectors. Various markers that may be used areknown in the art, and may include hprt, neomycin resistance, thymidinekinase, hygromycin resistance, etc. As used herein, the terms “cell,”“cell line,” and “cell culture” may be used interchangeably. In someembodiments, the host cell an immune cell or precursor thereof, e.g., aT cell, an NK cell, or an NKT cell. In some embodiments, one or more ofthese steps is performed at modules 208 of modular biological foundrysystem 200 by utilizing rigid cartridge 1100 and a robot materialtransfer system.

The present invention also provides genetically engineered cells whichinclude and stably express a subject CAR, dominant negative receptorand/or switch receptor, and/or bispecific antibody, and/or combinationsthereof, of the present disclosure. In some embodiments, the geneticallyengineered cells are genetically engineered T-lymphocytes (T cells),naive T cells (TN), memory T cells (for example, central memory T cells(TCM), effector memory cells (TEM)), natural killer cells (NK cells),and macrophages capable of giving rise to therapeutically relevantprogeny. In one embodiment, the genetically engineered cells areautologous cells.

In some embodiments, modified cells (e.g., including a subject CAR,dominant negative receptor and/or switch receptor, and/or expresses andsecretes a bispecific antibody, and/or combinations thereof) is producedby stably transfecting host cells with an expression vector including anucleic acid of the present disclosure. Additional methods to generate amodified cell of the present disclosure include, without limitation,chemical transformation methods (e.g., using calcium phosphate,dendrimers, liposomes and/or cationic polymers), non-chemicaltransformation methods (e.g., electroporation, optical transformation,gene electrotransfer and/or hydrodynamic delivery) and/or particle-basedmethods (e.g., impalefection, using a gene gun and/or magnetofection).Transfected cells expressing a subject CAR, dominant negative receptorand/or switch receptor, and/or bispecific antibody, and/or combinationsthereof, of the present disclosure may be expanded ex vivo.

Physical methods for introducing an expression vector into host cellsinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells including vectors and/or exogenous nucleic acids arewell-known in the art. See, Sambrook et al, 2001, “Molecular Cloning: ALaboratory Manual,” Cold Spring Harbor Laboratory, print, which ishereby incorporated by reference in its entirety. Chemical methods forintroducing an expression vector into a host cell include colloidaldispersion systems, such as macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes.

In some embodiments, lipids suitable for use in the manufacture of acellular engineering target at a modular biological foundry system isobtained from commercial sources. Stock solutions of lipids inchloroform or chloroform/methanol can be stored at about −20 degrees C.Chloroform may be used as the only solvent since it is more readilyevaporated than methanol. “Liposome” is a generic term encompassing avariety of single and multilamellar lipid vehicles formed by thegeneration of enclosed lipid bilayers or aggregates. Liposomes can becharacterized as having vesicular structures with a phospholipid bilayermembrane and an inner aqueous medium. Multilamellar liposomes havemultiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers. Compositions that have different structuresin solution than the normal vesicular structure are also encompassed.For example, the lipids may assume a micellar structure or merely existas non-uniform aggregates of lipid molecules. Also contemplated arelipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the nucleic acids in thehost cell, a variety of assays may be performed. Such assays include,for example, molecular biology assays well known to those of skill inthe art, such as Southern and Northern blotting, RT-PCR and PCR;biochemistry assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention. In some embodiments, one or more of theseassays is performed at modules 208 of modular biological foundry system200 by utilizing rigid cartridge 1100 and a robot material transfersystem.

In one embodiment, the nucleic acids introduced into the host cell areRNA. In another embodiment, the RNA is mRNA that comprises in vitrotranscribed RNA or synthetic RNA. The RNA may be produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA may be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA.

PCR may be used to generate a template for in vitro transcription ofmRNA which is then introduced into cells. Methods for performing PCR arewell known in the art. Primers for use in PCR are designed to haveregions that are substantially complementary to regions of the DNA to beused as a template for the PCR. “Substantially complementary,” as usedherein, refers to sequences of nucleotides where a majority or all ofthe bases in the primer sequence are complementary. Substantiallycomplementary sequences are able to anneal or hybridize with theintended DNA target under annealing conditions used for PCR. The primerscan be designed to be substantially complementary to any portion of theDNA template. For example, the primers can be designed to amplify theportion of a gene that is normally transcribed in cells (the openreading frame), including 5′ and 3′ UTRs. The primers may also bedesigned to amplify a portion of a gene that encodes a particular domainof interest. In one embodiment, the primers are designed to amplify thecoding region of a human cDNA, including all or portions of the 5′ and3′ UTRs. Primers useful for PCR are generated by synthetic methods thatare well known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

In some embodiments, chemical structures that have the ability topromote stability and/or translation efficiency of the RNA are used. TheRNA preferably has 5′ and 3′ UTRs. In one embodiment, the 5′ UTR isbetween zero and 3000 nucleotides in length. The length of 5′ and 3′ UTRsequences to be added to the coding region can be altered by differentmethods, including, but not limited to, designing primers for PCR thatanneal to different regions of the UTRs. Using this approach, one ofordinary skill in the art can modify the 5′ and 3′ UTR lengths requiredto achieve optimal translation efficiency following transfection of thetranscribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one embodiment, the promoter is a T7polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In one embodiment, the mRNA has both a cap on the 5′ end and a 3′poly(A) tail which determine ribosome binding, initiation of translationand stability mRNA in the cell. On a circular DNA template, forinstance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art.

In some embodiments, the RNA is electroporated into the cells, such asin vitro transcribed RNA. Any solutes suitable for cell electroporation,which can contain factors facilitating cellular permeability andviability such as sugars, peptides, lipids, proteins, antioxidants, andsurfactants can be included.

In some embodiments, a nucleic acid encoding a subject CAR, dominantnegative receptor and/or switch receptor, and/or bispecific antibody,and/or combinations thereof, of the present disclosure will be RNA,e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNAare known in the art; any known method can be used to synthesize RNAcomprising a sequence encoding a subject CAR, dominant negative receptorand/or switch receptor, and/or bispecific antibody, and/or combinationsthereof. Methods for introducing RNA into a host cell are known in theart. Introducing RNA comprising a nucleotide sequence encoding a subjectCAR, dominant negative receptor and/or switch receptor, and/orbispecific antibody, and/or combinations thereof, into a host cell canbe carried out in vitro or ex vivo or in vivo. For example, a host cell(e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporatedin vitro or ex vivo with RNA comprising a nucleotide sequence encoding asubject CAR, dominant negative receptor and/or switch receptor, and/orbispecific antibody, and/or combinations thereof.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the mRNAs with different structures andcombination of their domains.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free. A RNA transgenecan be delivered to a lymphocyte and expressed therein following a briefin vitro cell activation, as a minimal expressing cassette without theneed for any additional viral sequences. Under these conditions,integration of the transgene into the host cell genome is unlikely.Cloning of cells is not necessary because of the efficiency oftransfection of the RNA and its ability to uniformly modify the entirelymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation. Itis desirable to stabilize IVT-RNA using various modifications in orderto achieve prolonged expression of transferred IVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

In another aspect, the RNA construct is delivered into the cells byelectroporation. The various parameters including electric fieldstrength required for electroporation of any known cell type aregenerally known in the relevant research literature as well as numerouspatents and applications in the field. Electroporation may also beutilized to deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

In some embodiments, the immune cells (e.g. T cells) can be incubated orcultivated prior to, during and/or subsequent to introducing the nucleicacid molecule encoding the subject CAR, dominant negative receptorand/or switch receptor, and/or bispecific antibody, and/or combinationsthereof. In some embodiments, the cells (e.g. T cells) can be incubatedor cultivated prior to, during or subsequent to the introduction of thenucleic acid molecule encoding the subject CAR, dominant negativereceptor and/or switch receptor, and/or bispecific antibody, and/orcombinations thereof, such as prior to, during or subsequent to thetransduction of the cells with a viral vector (e.g. lentiviral vector)encoding the subject CAR, dominant negative receptor and/or switchreceptor, and/or bispecific antibody, and/or combinations thereof. Insome embodiments, the method includes activating or stimulating cellswith a stimulating or activating agent (e.g. anti-CD3/anti-CD28antibodies) prior to introducing the nucleic acid molecule encoding thesubject CAR, dominant negative receptor and/or switch receptor, and/orbispecific antibody, and/or combinations thereof. In some embodiments,one or more of these steps is performed at modules 208 of modularbiological foundry system 200 by utilizing rigid cartridge 1100 and arobot material transfer system.

In some embodiments, where the nucleic acid sequences encoding thesubject CAR, dominant negative receptor and/or switch receptor, and/orbispecific antibody, and/or combinations thereof, of the presentinvention reside on one or more separate nucleic acid sequences, theorder of introducing each of the one or more nucleic acid sequences mayvary. For example, a nucleic acid sequence encoding a subject CAR anddominant negative receptor and/or switch receptor may first beintroduced into the host cell, followed by introduction of a nucleicacid sequence encoding a subject bispecific antibody. For example, anucleic acid sequence encoding a subject bispecific antibody may firstbe introduced into the host cell, followed by introduction of a nucleicacid sequence encoding a subject CAR and dominant negative receptorand/or switch receptor. In some embodiments, each of the one or morenucleic acid sequences are introduced into the host cell simultaneously.Those of skill in the art will be able to determine the order in whicheach of the one or more nucleic acid sequences are introduced into thehost cell.

Prior to expansion, a source of immune cells is obtained from a subjectfor ex vivo manipulation. Sources of target cells for ex vivomanipulation may also include, e.g., autologous or heterologous donorblood, cord blood, or bone marrow. For example, the source of immunecells may be from the subject to be treated with the modified immunecells of the invention, e.g., the subject's blood, the subject's cordblood, or the subject's bone marrow. Non-limiting examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.Preferably, the subject is a human.

Immune cells can be obtained from a number of sources, including blood,peripheral blood mononuclear cells, bone marrow, lymph node tissue,spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cellsare cells of the immune system, such as cells of the innate or adaptiveimmunity, e.g., myeloid or lymphoid cells, including lymphocytes,typically T cells and/or NK cells. Other exemplary cells include stemcells, such as multipotent and pluripotent stem cells, including inducedpluripotent stem cells (iPSCs). In some embodiments, the cells are humancells. With reference to the subject to be treated, the cells may beallogeneic and/or autologous. The cells typically are primary cells,such as those isolated directly from a subject and/or isolated from asubject and frozen.

In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell(e.g., a CD8+ naive T cell, central memory T cell, or effector memory Tcell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatoryT cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell ahematopoietic stem cell, a natural killer cell (NK cell) or a dendriticcell. In some embodiments, the cells are monocytes or granulocytes,e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mastcells, eosinophils, and/or basophils. In an embodiment, the target cellis an induced pluripotent stem (iPS) cell or a cell derived from an iPScell, e.g., an iPS cell generated from a subject, manipulated to alter(e.g., induce a mutation in) or manipulate the expression of one or moretarget genes, and differentiated into, e.g., a T cell, e.g., a CD8+ Tcell (e.g., a CD8+ naive T cell, central memory T cell, or effectormemory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoidprogenitor cell or a hematopoietic stem cell.

In some embodiments, the cells include one or more subsets of T cells orother cell types, such as whole T cell populations, CD4+ cells, CD8+cells, and subpopulations thereof, such as those defined by function,activation state, maturity, potential for differentiation, expansion,recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. Among the sub-types and subpopulations of Tcells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells,effector T cells (TEFF), memory T cells and sub-types thereof, such asstem cell memory T (TSCM), central memory T (TCM), effector memory T(TEM), or terminally differentiated effector memory T cells,tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells,helper T cells, cytotoxic T cells, mucosa-associated invariant T (MATT)cells, naturally occurring and adaptive regulatory T (Treg) cells,helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9cells, TH22 cells, follicular helper T cells, alpha/beta T cells, anddelta/gamma T cells. In certain embodiments, any number of T cell linesavailable in the art, may be used.

In some embodiments, the methods include isolating immune cells from thesubject, preparing, processing, culturing, and/or engineering them. Insome embodiments, preparation of the engineered cells includes one ormore culture and/or preparation steps. The cells for engineering asdescribed may be isolated from a sample, such as a biological sample,e.g., one obtained from or derived from a subject. In some embodiments,the subject from which the cell is isolated is one having the disease orcondition or in need of a cell therapy or to which cell therapy will beadministered. The subject in some embodiments is a human in need of aparticular therapeutic intervention, such as the adoptive cell therapyfor which cells are being isolated, processed, and/or engineered.Accordingly, the cells in some embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In some embodiments, the sample from which the cells are derived orisolated is blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig. Insome embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components. In someembodiments, one or more of these steps is performed at modules 208 ofmodular biological foundry system 200 by utilizing rigid cartridge 1100and a robot material transfer system.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someembodiments, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and, in some embodiments, contains cells otherthan red blood cells and platelets. In some embodiments, the blood cellscollected from the subject are washed, e.g., to remove the plasmafraction and to place the cells in an appropriate buffer or media forsubsequent processing steps. In some embodiments, the cells are washedwith phosphate buffered saline (PBS). In some embodiments, a washingstep is accomplished by tangential flow filtration (TFF) according tothe manufacturer's instructions. In some embodiments, the cells areresuspended in a variety of biocompatible buffers after washing. Incertain embodiments, components of a blood cell sample are removed andthe cells directly resuspended in culture media. In some embodiments,the methods include density-based cell separation methods, such as thepreparation of white blood cells from peripheral blood by lysing the redblood cells and centrifugation through a Percoll or Ficoll gradient. Insome embodiments, one or more of these steps is performed at modules 208of modular biological foundry system 200 by utilizing rigid cartridge1100 and a robot material transfer system.

In one embodiment, immune cells are obtained cells from the circulatingblood of an individual are obtained by apheresis or leukapheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. The cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media, such as phosphate buffered saline (PBS) orwash solution lacks calcium and may lack magnesium or may lack many ifnot all divalent cations, for subsequent processing steps. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample are removed and the cellsdirectly resuspended in culture media. In some embodiments, one or moreof these steps is performed at modules 208 of modular biological foundrysystem 200 by utilizing rigid cartridge 1100 and a robot materialtransfer system.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner. In some embodiments, one or more of these steps is performed atmodules 208 of modular biological foundry system 200 by utilizing rigidcartridge 1100 and a robot material transfer system.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some embodiments, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population. The separation need not result in 100%enrichment or removal of a particular cell population or cellsexpressing a particular marker. For example, positive selection of orenrichment for cells of a particular type, such as those expressing amarker, refers to increasing the number or percentage of such cells, butneed not result in a complete absence of cells not expressing themarker. Likewise, negative selection, removal, or depletion of cells ofa particular type, such as those expressing a marker, refers todecreasing the number or percentage of such cells, but need not resultin a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

In some embodiments, one or more of the T cell populations is enrichedfor or depleted of cells that are positive for (marker+) or express highlevels (marker.sup.high) of one or more particular markers, such assurface markers, or that are negative for (marker.sup.−) or expressrelatively low levels (marker.sup.low) of one or more markers. Forexample, in some embodiments, specific subpopulations of T cells, suchas cells positive or expressing high levels of one or more surfacemarkers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+,and/or CD45RO+ T cells, are isolated by positive or negative selectiontechniques. In some cases, such markers are those that are absent orexpressed at relatively low levels on certain populations of T cells(such as non-memory cells) but are present or expressed at relativelyhigher levels on certain other populations of T cells (such as memorycells). In one embodiment, the cells (such as the CD8+ cells or the Tcells, e.g., CD3+ cells) are enriched for (i.e., positively selectedfor) cells that are positive or expressing high surface levels ofCD45RO, CCR7, CD28, CD27, CD44, CD 127, and/or CD62L and/or depleted of(e.g., negatively selected for) cells that are positive for or expresshigh surface levels of CD45RA. In some embodiments, cells are enrichedfor or depleted of cells positive or expressing high surface levels ofCD 122, CD95, CD25, CD27, and/or IL7-Ra (CD 127). In some examples, CD8+T cells are enriched for cells positive for CD45RO (or negative forCD45RA) and for CD62L. For example, CD3+, CD28+ T cells can bepositively selected using CD3/CD28 conjugated magnetic beads (e.g.,DYNABEADS® M-450 CD3/CD28 T Cell Expander).

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In someembodiments, a CD4+ or CD8+ selection step is used to separate CD4+helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can befurther sorted into sub-populations by positive or negative selectionfor markers expressed or expressed to a relatively higher degree on oneor more naive, memory, and/or effector T cell subpopulations. In someembodiments, CD8+ cells are further enriched for or depleted of naive,central memory, effector memory, and/or central memory stem cells, suchas by positive or negative selection based on surface antigensassociated with the respective subpopulation. In some embodiments,enrichment for central memory T (TCM) cells is carried out to increaseefficacy, such as to improve long-term survival, expansion, and/orengraftment following administration, which, in some embodiments, isparticularly robust in such sub-populations. In some embodiments,combining TCM-enriched CD8+ T cells and CD4+ T cells further enhancesefficacy.

In embodiments, memory T cells are present in both CD62L+ and CD62L−subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as usinganti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cellpopulation and a CD8+ T cell sub-population, e.g., a sub-populationenriched for central memory (TCM) cells. In some embodiments, theenrichment for central memory T (TCM) cells is based on positive or highsurface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; insome embodiments, it is based on negative selection for cells expressingor highly expressing CD45RA and/or granzyme B. In some embodiments,isolation of a CD8+ population enriched for TCM cells is carried out bydepletion of cells expressing CD4, CD 14, CD45RA, and positive selectionor enrichment for cells expressing CD62L. In one aspect, enrichment forcentral memory T (TCM) cells is carried out starting with a negativefraction of cells selected based on CD4 expression, which is subjectedto a negative selection based on expression of CD 14 and CD45RA, and apositive selection based on CD62L. Such selections, in some embodiments,are carried out simultaneously and in other aspects are carried outsequentially, in either order. In some embodiments, the same CD4expression-based selection step used in preparing the CD8+ cellpopulation or subpopulation, also is used to generate the CD4+ cellpopulation or sub-population, such that both the positive and negativefractions from the CD4-based separation are retained and used insubsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

CD4+ T helper cells are sorted into naive, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4+ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+T cells. In some embodiments, central memory CD4+ cells are CD62L+ andCD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO.In one example, to enrich for CD4+ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Insome embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor. The conditions caninclude one or more of particular media, temperature, oxygen content,carbon dioxide content, time, agents, e.g., nutrients, amino acids,antibiotics, ions, and/or stimulatory factors, such as cytokines,chemokines, antigens, binding partners, fusion proteins, recombinantsoluble receptors, and any other agents designed to activate the cells.In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some embodiments,the agent turns on or initiates TCR/CD3 intracellular signaling cascadein a T cell. Such agents can include antibodies, such as those specificfor a TCR component and/or costimulatory receptor, e.g., anti-CD3,anti-CD28, for example, bound to solid support such as a bead, and/orone or more cytokines. Optionally, the expansion method may furthercomprise the step of adding anti-CD3 and/or anti CD28 antibody to theculture medium (e.g., at a concentration of at least about 0.5 ng/ml).In some embodiments, the stimulating agents include IL-2 and/or IL-15,for example, an IL-2 concentration of at least about 10 units/mL. Insome embodiments, one or more of these steps is performed at modules 208of modular biological foundry system 200 by utilizing rigid cartridge1100 and a robot material transfer system.

In another embodiment, T cells are isolated from peripheral blood bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient. Alternatively, T cells canbe isolated from an umbilical cord. In any event, a specificsubpopulation of T cells can be further isolated by positive or negativeselection techniques. In some embodiments, one or more of these steps isperformed at modules 208 of modular biological foundry system 200 byutilizing rigid cartridge 1100 and a robot material transfer system.

The cord blood mononuclear cells so isolated can be depleted of cellsexpressing certain antigens, including, but not limited to, CD34, CD8,CD14, CD19, and CD56. Depletion of these cells can be accomplished usingan isolated antibody, a biological sample comprising an antibody, suchas ascites, an antibody bound to a physical support, and a cell boundantibody.

Enrichment of a T cell population by negative selection can beaccomplished using a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immuno-adherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4.sup.+ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. In some embodiments, one or more ofthese steps is performed at modules 208 of modular biological foundrysystem 200 by utilizing rigid cartridge 1100 and a robot materialtransfer system.

T cells can also be frozen after the washing step, which does notrequire the monocyte-removal step. While not wishing to be bound bytheory, the freeze and subsequent thaw step provides a more uniformproduct by removing granulocytes and to some extent monocytes in thecell population. After the washing step that removes plasma andplatelets, the cells may be suspended in a freezing solution. While manyfreezing solutions and parameters are known in the art and will beuseful in this context, in a non-limiting example, one method involvesusing PBS containing 20% DMSO and 8% human serum albumin, or othersuitable cell freezing media. The cells are then frozen to −80 degreesC. at a rate of 1 degrees C. per minute and stored in the vapor phase ofa liquid nitrogen storage tank. Other methods of controlled freezing maybe used as well as uncontrolled freezing immediately at −20 degrees C.or in liquid nitrogen. In some embodiments, one or more of these stepsis performed at modules 208 of modular biological foundry system 200 byutilizing rigid cartridge 1100 and a robot material transfer system.

In one embodiment, the population of T cells includes cells such asperipheral blood mononuclear cells, cord blood cells, a purifiedpopulation of T cells, and a T cell line. In another embodiment,peripheral blood mononuclear cells include the population of T cells. Inyet another embodiment, purified T cells include the population of Tcells.

In certain embodiments, T regulatory cells (Tregs) is isolated from asample. The sample can include, but is not limited to, umbilical cordblood or peripheral blood. In certain embodiments, the Tregs areisolated by flow-cytometry sorting. The sample can be enriched for Tregsprior to isolation by any means known in the art. The isolated Tregs canbe cryopreserved, and/or expanded prior to use. In some embodiments, oneor more of these steps is performed at modules 208 of modular biologicalfoundry system 200 by utilizing rigid cartridge 1100 and a robotmaterial transfer system.

Whether prior to or after modification of cells to express a subjectCAR, dominant negative receptor, and/or switch receptor, and/orbispecific antibody, and/or combinations thereof, the cells can beactivated and expanded in number using methods known to one of skill inthe art. For example, the T cells of the invention may be expanded bycontact with a surface having attached thereto an agent that stimulatesa CD3/TCR complex associated signal and a ligand that stimulates aco-stimulatory molecule on the surface of the T cells. In particular, Tcell populations may be stimulated by contact with an anti-CD3 antibody,or an antigen-binding fragment thereof, or an anti-CD2 antibodyimmobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,T cells can be contacted with an anti-CD3 antibody and an anti-CD28antibody, under conditions appropriate for stimulating proliferation ofthe T cells. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 and these can be used in the present disclosure as can othermethods and reagents known in the art.

Expanding T cells by the methods disclosed herein can be multiplied byabout 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold,4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and anyand all whole or partial integers therebetween. In one embodiment, the Tcells expand in the range of about 20-fold to about 50-fold.

Following culturing, the T cells can be incubated in cell medium in aculture apparatus for a period of time or until the cells reachconfluency or high cell density for optimal passage before passing thecells to another culture apparatus. The culturing apparatus can be ofany culture apparatus commonly used for culturing cells in vitro.Preferably, the level of confluence is 70% or greater before passing thecells to another culture apparatus. More preferably, the level ofconfluence is 90% or greater. A period of time can be any time suitablefor the culture of cells in vitro. The T cell medium may be replacedduring the culture of the T cells at any time. Preferably, the T cellmedium is replaced about every 2 to 3 days. The T cells are thenharvested from the culture apparatus whereupon the T cells can be usedimmediately or cryopreserved to be stored for use at a later time. Inone embodiment, the invention includes cryopreserving the expanded Tcells. The cryopreserved T cells are thawed prior to introducing nucleicacids into the T cell.

In another embodiment, the method comprises isolating T cells andexpanding the T cells. In another embodiment, the invention furthercomprises cryopreserving the T cells prior to expansion. In yet anotherembodiment, the cryopreserved T cells are thawed for electroporationwith the RNA encoding the chimeric membrane protein. In someembodiments, one or more of these steps is performed at modules 208 ofmodular biological foundry system 200 by utilizing rigid cartridge 1100and a robot material transfer system.

The culturing step as described herein (contact with agents as describedherein or after electroporation) can be very short, for example lessthan 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as describedfurther herein (contact with agents as described herein) can be longer,for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition. A primary cell culture is a culture of cells,tissues or organs taken directly from an organism and before the firstsubculture. Cells are expanded in culture when they are placed in agrowth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is typicallymeasured by the amount of time required for the cells to double innumber, otherwise known as the doubling time. In some embodiments, oneor more of these steps is performed at modules 208 of modular biologicalfoundry system 200 by utilizing rigid cartridge 1100 and a robotmaterial transfer system.

Each round of subculturing is referred to as a passage. When cells aresubcultured, they are referred to as having been passaged. A specificpopulation of cells, or a cell line, is sometimes referred to orcharacterized by the number of times it has been passaged. For example,a cultured cell population that has been passaged ten times may bereferred to as a P10 culture. The primary culture, i.e., the firstculture following the isolation of cells from tissue, is designated P0.Following the first subculture, the cells are described as a secondaryculture (P1 or passage 1). After the second subculture, the cells becomea tertiary culture (P2 or passage 2), and so on. It will be understoodby those of skill in the art that there may be many population doublingsduring the period of passaging. Therefore, the number of populationdoublings of a culture is greater than the passage number. The expansionof cells (i.e., the number of population doublings) during the periodbetween passaging depends on many factors, including but is not limitedto the seeding density, substrate, medium, and time between passaging.In some embodiments, one or more of these steps is performed at modules208 of modular biological foundry system 200 by utilizing rigidcartridge 1100 and a robot material transfer system.

In one embodiment, the cells may be cultured for several hours (about 3hours) to about 14 days or any hourly integer value in between.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10,IL-12, IL-15, TGF-beta, and TNF-.alpha. or any other additives for thegrowth of cells known to the skilled artisan. Other additives for thegrowth of cells include, but are not limited to, surfactant, plasmanate,and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.Media can include RPMI 1640, AIM-V, DMEM, MEM, .alpha.-MEM, F-12, X-Vivo15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate,and vitamins, either serum-free or supplemented with an appropriateamount of serum (or plasma) or a defined set of hormones, and/or anamount of cytokine(s) sufficient for the growth and expansion of Tcells. Antibiotics, e.g., penicillin and streptomycin, are included onlyin experimental cultures, not in cultures of cells that are to beinfused into a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37 degrees C.) and atmosphere (e.g., air plus 5% CO.sub.2).

The medium used to culture the T cells may include an agent that canco-stimulate the T cells. For example, an agent that can stimulate CD3is an antibody to CD3, and an agent that can stimulate CD28 is anantibody to CD28. A cell isolated by the methods disclosed herein can beexpanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold,500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold,3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, orgreater. In one embodiment, the T cells expand in the range of about20-fold to about 50-fold, or more. In one embodiment, human T regulatorycells are expanded via anti-CD3 antibody coated KT64.86 artificialantigen presenting cells (aAPCs). In one embodiment, human T regulatorycells are expanded via anti-CD3 antibody coated K562 artificial antigenpresenting cells (aAPCs). In some embodiments, one or more of thesesteps is performed at modules 208 of modular biological foundry system200 by utilizing rigid cartridge 1100 and a robot material transfersystem.

In one embodiment, the method of expanding the T cells can furthercomprise isolating the expanded T cells for further applications. Inanother embodiment, the method of expanding can further comprise asubsequent electroporation of the expanded T cells followed byculturing. The subsequent electroporation may include introducing anucleic acid encoding an agent, such as transducing the expanded Tcells, transfecting the expanded T cells, or electroporating theexpanded T cells with a nucleic acid, into the expanded population of Tcells, wherein the agent further stimulates the T cell. The agent maystimulate the T cells, such as by stimulating further expansion,effector function, or another T cell function. In some embodiments, oneor more of these steps is performed at modules 208 of modular biologicalfoundry system 200 by utilizing rigid cartridge 1100 and a robotmaterial transfer system.

Example 1: Methods of Treatment of a Subject Using Cellular EngineeringTargets

In some embodiments, the cellular engineering targets is modified cells(e.g., T cells). In some embodiments, a composition for immunotherapyincludes the modified cells. In some embodiments, the compositionincludes a pharmaceutical composition and further include apharmaceutically acceptable carrier. In some embodiments, atherapeutically effective amount of the pharmaceutical compositioninclude the modified T cells is administered.

In one aspect, the present disclosure includes a method for adoptivecell transfer therapy including administering to a subject in needthereof a cellular engineering target including a modified T cell of thepresent disclosure. In another aspect, the present disclosure includes amethod of treating a disease or condition in a subject includingadministering to a subject in need thereof a population of modified Tcells

In some embodiments, a method of treating a disease or condition in asubject in need thereof includes administering to the subject a modifiedcell (e.g., modified T cell) of the present invention. In oneembodiment, the method of treating a disease or condition in a subjectin need thereof comprises administering to the subject a modified cell(e.g., a modified T cell) comprising a subject CAR, dominant negativereceptor and/or switch receptor, and/or a bispecific antibody, and/orcombinations thereof. In one embodiment, the method of treating adisease or condition in a subject in need thereof comprisesadministering to the subject a modified cell (e.g., a modified T cell)comprising a subject CAR (e.g., a CAR having affinity for PSMA on atarget cell) and a dominant negative receptor and/or switch receptor. Inone embodiment, the method of treating a disease or condition in asubject in need thereof comprises administering to the subject amodified cell (e.g., a modified T cell) comprising a subject CAR (e.g.,a CAR having affinity for PSMA on a target cell), a dominant negativereceptor and/or switch receptor, and wherein the modified cell iscapable of expressing and secreting a bispecific antibody.

Methods for administration of immune cells for adoptive cell therapy areknown and may be used in connection with the provided methods andcompositions. In some embodiments, autologous transfer conducts the celltherapy, e.g., adoptive T cell therapy, in which the cells are isolatedand/or otherwise prepared from the subject who is to receive the celltherapy, or from a sample derived from such a subject. Thus, in someembodiments, the cells are derived from a subject, e.g., patient, inneed of a treatment and the cells, following isolation and processingare administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by allogeneic transfer, by isolating and/or otherwisepreparing the cells from a subject other than a subject who is toreceive or who ultimately receives the cell therapy, e.g., a firstsubject. In such embodiments, the method includes administering to adifferent subject, e.g., a second subject, of the same species. In someembodiments, the first and second subjects are genetically identical. Insome embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class orsupertype as the first subject.

In some embodiments, the method includes treating the subject with atherapeutic agent targeting the disease or condition, e.g., the tumor,prior to administering of the cells or composition containing the cells.In some embodiments, the subject is refractory or non-responsive to theother therapeutic agent. In some embodiments, the subject has persistentor relapsed disease, e.g., following treatment with another therapeuticintervention, including chemotherapy, radiation, and/or hematopoieticstem cell transplantation (HSCT), e.g., allogenic HSCT. In someembodiments, the administering the cellular engineering targeteffectively treats the subject despite the subject having becomeresistant to another therapy.

In some embodiments, the subject is responsive to the other therapeuticagent, and treatment with the therapeutic agent reduces disease burden.In some embodiments, the subject is initially responsive to thetherapeutic agent, but exhibits a relapse of the disease or conditionover time. In some embodiments, the subject has not relapsed. In somesuch embodiments, a determination that the subject is at risk forrelapse is provided, such as at a high risk of relapse, and thus themethod includes administering cellular engineering targetprophylactically, e.g., to reduce the likelihood of or prevent relapse.In some embodiments, the subject has not received prior treatment withanother therapeutic agent.

In some embodiments, the subject has persistent or relapsed disease,e.g., following treatment with another therapeutic intervention,including chemotherapy, radiation, and/or hematopoietic stem celltransplantation (HSCT), e.g., allogenic HSCT. In some embodiments, theadministrating the cellular engineering target effectively treats thesubject despite the subject having become resistant to another therapy.

In some embodiments, the method includes administering the modifiedimmune cells of the cellular engineering target of the presentdisclosure to an animal, preferably a mammal, even more preferably ahuman, to treat a cancer. In addition, in some embodiments, the cellularengineering target of the present invention is utilized for thetreatment of any condition related to a cancer, especially acell-mediated immune response against a tumor cell(s), where it isdesirable to treat or alleviate the disease. The types of cancers withthe modified cells or pharmaceutical compositions of the inventioninclude, carcinoma, blastoma, and sarcoma, and certain leukemia orlymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Other exemplary cancersinclude but are not limited breast cancer, prostate cancer, ovariancancer, cervical cancer, skin cancer, pancreatic cancer, colorectalcancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia,lung cancer, thyroid cancer, and the like. The cancers may be non-solidtumors (such as hematological tumors) or solid tumors. Adulttumors/cancers and pediatric tumors/cancers are also included.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In some embodiments, carcinomas amenable to therapy by a methoddisclosed herein include, but are not limited to, esophageal carcinoma,hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer),squamous cell carcinoma (various tissues), bladder carcinoma, includingtransitional cell carcinoma (a malignant neoplasm of the bladder),bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastriccarcinoma, lung carcinoma, including small cell carcinoma and non-smallcell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma,pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterinecarcinoma, testicular carcinoma, osteogenic carcinoma, epithelialcarcinoma, and nasopharyngeal carcinoma.

In some embodiments, sarcomas amenable to therapy by a method disclosedherein include, but are not limited to, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma,leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Prostate adenocarcinoma is an extremely common and lethal disease.Prostate cancer is the most common malignancy among men. Prostate canceris the second-leading cause of cancer-related deaths among men,accounting for an estimated 10% of annual male cancer deaths. PSMA ishighly expressed in malignant prostate tissue, with low-levels ofexpression in some normal human tissues. Under normal physiologicconditions, PSMA is expressed in the prostate gland (secretory acinarepithelium), kidney (proximal tubules), nervous system glia (astrocytesand Schwann cells), and the small intestine (jejunal brush border). PSMAis much more highly expressed in prostate epithelium and issignificantly unregulated in malignant prostate tissues. PSMA expressionin normal cells has been found to be 100-fold to 1000-fold less than inprostate carcinoma cells. PSMA expression increases significantly duringthe transformation from benign prostatic hyperplasia to prostaticadenocarcinoma. PSMA expression has been found to be directly correlatedwith the histologic grade of malignant prostate tissue and increaseswith more advanced disease (i.e. highest PSMA expression found inprostate cancer metastases in lymph node and bone).

In one embodiment, the methods of the invention are useful for treatingprostate cancer, for example advanced castrate-resistant prostatecancer. It should be readily understood by one of ordinary skill in theart that any type of cancer wherein the PSMA tumor antigen is expressed,can be treated using the methods of the present invention. For example,neovascular expression of PSMA was found in non-small cell lung cancer.Accordingly, the methods of the invention may also be useful fortreating non-small cell lung cancer (NSCLC).

In certain exemplary embodiments, the modified immune cells of theinvention treat prostate cancer. In one embodiment, a method of thepresent disclosure provides a treatment for castrate-resistant prostatecancer. In one embodiment, a method of the present disclosure provides atreatment for advanced castrate-resistant prostate cancer. In oneembodiment, a method of the present disclosure provides a treatment formetastatic castrate-resistant prostate cancer. In one embodiment, amethod of the present disclosure provides a treatment for metastaticcastrate-resistant prostate cancer, wherein the patient with metastaticcastrate-resistant prostate cancer has .gtoreq.10% tumor cellsexpressing PSMA. In one embodiment, a method of the present disclosureprovides a treatment for castrate-resistant prostate adenocarcinoma,wherein the patient has castrate levels of testosterone (e.g., <50ng/mL) with or without the use of androgen deprivation therapy.

In certain embodiments, the method includes providing the subject with asecondary treatment. Secondary treatments include but are not limited tochemotherapy, radiation, surgery, and medications.

In some embodiments, the method includes administering the cellularengineering in dosages and routes and at times determined based onappropriate pre-clinical and clinical experimentation and trials. Insome embodiments, the method includes administering cellular engineeringtarget compositions multiple times at dosages within these ranges. Theadministrating of the cells of the invention includes other methodsuseful to treat the desired disease or condition as determined by thoseof skill in the art.

In some embodiments, administrating of the cellular engineering targetof the present disclosure includes any convenient manner known to thoseof skill in the art. In some embodiments, administrating of the cellularengineering target includes aerosol inhalation, injection, ingestion,transfusion, implantation or transplantation. In some embodiments,administrating of the cellular engineering target compositions includestransarterially, subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous (i.v.)injection, or intraperitoneally. In some embodiments, administrating ofthe cellular engineering target includes injection into a site of thesubject, a local disease site in the subject, alymph node, an organ, atumor, and the like.

In some embodiments, administrating of the cellular engineering targetis at a desired dosage, which includes a desired dose or number of cellsor cell type(s) and/or a desired ratio of cell types. Thus, the dosageof cells in some embodiments is based on a total number of cells (ornumber per kg body weight) and a desired ratio of the individualpopulations or sub-types, such as the CD4+ to CD8+ ratio. In someembodiments, the dosage of cells is based on a desired total number (ornumber per kg of body weight) of cells in the individual populations orof individual cell types. In some embodiments, the dosage is based on acombination of such features, such as a desired number of total cells,desired ratio, and desired total number of cells in the individualpopulations.

In some embodiments, the method includes administering the populationsor sub-types of cells, such as CD8.sup.+ and CD4.sup.+ T cells, at orwithin a tolerated difference of a desired dose of total cells, such asa desired dose of T cells. In some embodiments, the desired dose is adesired number of cells or a desired number of cells per unit of bodyweight of the subject to whom the cells are administered, e.g.,cells/kg. In some embodiments, the desired dose is at or above a minimumnumber of cells or minimum number of cells per unit of body weight. Insome embodiments, among the total cells, the individual populations orsub-types are present at or near a desired output ratio (such asCD4.sup.+ to CD8.sup.+ ratio), e.g., within a certain tolerateddifference or error of such a ratio.

In some embodiments, the method includes administrating of the cellularengineering target at or within a tolerated difference of a desired doseof one or more of the individual populations or sub-types of cells, suchas a desired dose of CD4+ cells and/or a desired dose of CD8+ cells. Insome embodiments, the desired dose is a desired number of cells of thesub-type or population, or a desired number of such cells per unit ofbody weight of the subject to whom the cells are administered, e.g.,cells/kg. In some embodiments, the desired dose is at or above a minimumnumber of cells of the population or subtype, or minimum number of cellsof the population or sub-type per unit of body weight. Thus, in someembodiments, the dosage is based on a desired fixed dose of total cellsand a desired ratio, and/or based on a desired fixed dose of one ormore, e.g., each, of the individual sub-types or sub-populations. Thus,in some embodiments, the dosage is based on a desired fixed or minimumdose of T cells and a desired ratio of CD4.sup.+ to CD8.sup.+ cells,and/or is based on a desired fixed or minimum dose of CD4.sup.+ and/orCD8.sup.+ cells.

In some embodiments, the method includes administrating of the cellularengineering target to the subject at a range of about one million toabout 100 billion cells, such as, e.g., 1 million to about 50 billioncells (e.g., about 5 million cells, about 25 million cells, about 500million cells, about 1 billion cells, about 5 billion cells, about 20billion cells, about 30 billion cells, about 40 billion cells, or arange defined by any two of the foregoing values), such as about 10million to about 100 billion cells (e.g., about 20 million cells, about30 million cells, about 40 million cells, about 60 million cells, about70 million cells, about 80 million cells, about 90 million cells, about10 billion cells, about 25 billion cells, about 50 billion cells, about75 billion cells, about 90 billion cells, or a range defined by any twoof the foregoing values), and in some cases about 100 million cells toabout 50 billion cells (e.g., about 120 million cells, about 250 millioncells, about 350 million cells, about 450 million cells, about 650million cells, about 800 million cells, about 900 million cells, about 3billion cells, about 30 billion cells, about 45 billion cells) or anyvalue in between these ranges.

In some embodiments, the dose of total cells and/or dose of individualsub-populations of cells is within a range of between at or about1.times.10.sup.5 cells/kg to about 1.times.10.sup.11 cells/kg, 10.sup.4,and at or about 10.sup.11 cells/kilograms (kg) body weight, such asbetween 10.sup.5 and 10.sup.6 cells/kg body weight, for example, at orabout 1.times.10.sup.5 cells/kg, 1.5.times.10.sup.5 cells/kg,2.times.10.sup.5 cells/kg, or 1.times.10.sup.6 cells/kg body weight. Forexample, in some embodiments, the cells are administered at, or within acertain range of error of, between at or about 10.sup.4 and at or about10.sup.9 T cells/kilograms (kg) body weight, such as between 10.sup.5and 10.sup.6 T cells/kg body weight, for example, at or about1.times.10.sup.5 T cells/kg, 1.5.times.10.sup.5 T cells/kg,2.times.10.sup.5 T cells/kg, or 1.times.10.sup.6 T cells/kg body weight.In other exemplary embodiments, a suitable dosage range of modifiedcells for use in a method of the present disclosure includes, withoutlimitation, from about 1.times.10.sup.5 cells/kg to about1.times.10.sup.6 cells/kg, from about 1.times.10.sup.6 cells/kg to about1.times.10.sup.7 cells/kg, from about 1.times.10.sup.7 cells/kg about1.times.10.sup.8 cells/kg, from about 1.times.10.sup.8 cells/kg about1.times.10.sup.9 cells/kg, from about 1.times.10.sup.9 cells/kg about1.times.10.sup.10 cells/kg, from about 1.times.10.sup.10 cells/kg about1.times.10.sup.11 cells/kg. In an exemplary embodiment, a suitabledosage for use in a method of the present disclosure is about1.times.10.sup.8 cells/kg. In an exemplary embodiment, a suitable dosagefor use in a method of the present disclosure is about 1.times.10.sup.7cells/kg. In other embodiments, a suitable dosage is from about1.times.10.sup.7 total cells to about 5.times.10.sup.7 total cells. Insome embodiments, a suitable dosage is from about 1.times.10.sup.8 totalcells to about 5.times.10.sup.8 total cells. In some embodiments, asuitable dosage is from about 1.4.times.10.sup.7 total cells to about1.1.times.10.sup.9 total cells. In an exemplary embodiment, a suitabledosage for use in a method of the present disclosure is about7.times.10.sup.9 total cells. In an exemplary embodiment, a suitabledosage is from about 1.times.10.sup.7 total cells to about3.times.10.sup.7 total cells.

In some embodiments, the dose of total cells and/or dose of individualsub-populations of cells is within a range of between at or about1.times.10.sup.5 cells/m.sup.2 to about 1.times.10.sup.11 cells/m.sup.2.In an exemplary embodiment, the dose of total cells and/or dose ofindividual sub-populations of cells is within a range of between at orabout 1.times.10.sup.7/m.sup.2 to at or about 3.times.10.sup.7/m.sup.2.In an exemplary embodiment, the dose of total cells and/or dose ofindividual sub-populations of cells is within a range of between at orabout 1.times.10.sup.8/m.sup.2 to at or about 3.times.10.sup.8/m.sup.2.In some embodiments, the dose of total cells and/or dose of individualsub-populations of cells is the maximum tolerated dose by a givenpatient.

In some embodiments, the method includes administrating the cellularengineering target at or within a certain range of error of between ator about 10.sup.4 and at or about 10.sup.9 CD4.sup.+ and/or CD8.sup.+cells/kilograms (kg) body weight, such as between 10.sup.5 and 10.sup.6CD4.sup.+ and/or CD8.sup.+ cells/kg body weight, for example, at orabout 1.times.10.sup.5 CD4.sup.+ and/or CD8.sup.+ cells/kg,1.5.times.10.sup.5 CD4.sup.+ and/or CD8.sup.+ cells/kg, 2.times.10.sup.5CD4.sup.+ and/or CD8.sup.+ cells/kg, or 1.times.10.sup.6 CD4.sup.+and/or CD8.sup.+ cells/kg body weight. In some embodiments, the cellsare administered at or within a certain range of error of, greater than,and/or at least about 1.times.10.sup.6, about 2.5.times.10.sup.6, about5.times.10.sup.6, about 7.5.times.10.sup.6, or about 9.times.10.sup.6CD4.sup.+ cells, and/or at least about 1.times.10.sup.6, about2.5.times.10.sup.6, about 5.times.10.sup.6, about 7.5.times.10.sup.6, orabout 9.times.10.sup.6 CD8+ cells, and/or at least about1.times.10.sup.6, about 2.5.times.10.sup.6, about 5.times.10.sup.6,about 7.5.times.10.sup.6, or about 9.times.10.sup.6 T cells. In someembodiments, the cells are administered at or within a certain range oferror of between about 10.sup.8 and 10.sup.12 or between about 10.sup.10and 10.sup.11 T cells, between about 10.sup.8 and 10.sup.12 or betweenabout 10.sup.10 and 10.sup.11 CD4.sup.+ cells, and/or between about10.sup.8 and 10.sup.12 or between about 10.sup.10 and 10.sup.11CD8.sup.+ cells.

In some embodiments, the method includes administrating the cellularengineering target with a toleration range of a desired output ratio ofmultiple cell populations or sub-types, such as CD4+ and CD8+ cells orsub-types. In some embodiments, the desired ratio is a specific ratio orcan be a range of ratios, for example, in some embodiments, the desiredratio (e.g., ratio of CD4.sup.+ to CD8.sup.+ cells) is between at orabout 5:1 and at or about 5:1 (or greater than about 1:5 and less thanabout 5:1), or between at or about 1:3 and at or about 3:1 (or greaterthan about 1:3 and less than about 3:1), such as between at or about 2:1and at or about 1:5 (or greater than about 1:5 and less than about 2:1,such as at or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1,1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1,1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9:1:2, 1:2.5, 1:3,1:3.5, 1:4, 1:4.5, or 1:5. In some embodiments, the tolerated differenceis within about 1%, about 2%, about 3%, about 4% about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50% of the desired ratio, including any value in betweenthese ranges.

In some embodiments, the method includes administrating the cellularengineering target a dose of modified cells in a single dose or multipledoses. In some embodiments, administrating the cellular engineeringincludes multiple doses, e.g., once a week or every 7 days, once every 2weeks or every 14 days, once every 3 weeks or every 21 days, once every4 weeks or every 28 days. In an exemplary embodiment, administrating thecellular engineering includes a single dose of modified cells, such asby rapid intravenous infusion.

In some embodiments, for the prevention or treatment of disease, theappropriate dosage depends on the type of disease, the type of cells orrecombinant receptors, the severity and course of the disease, whetheradministrating the cells for preventive or therapeutic purposes,previous therapy, the subject's clinical history and response to thecellular engineering target, and the discretion of the attendingphysician. In some embodiments, the method includes administrating thecompositions and cells once or over a series of treatments.

In some embodiments, the cells are administered as part of a combinationtreatment, such as simultaneously with or sequentially with, in anyorder, another therapeutic intervention, such as an antibody orengineered cell or receptor or agent, such as a cytotoxic or therapeuticagent. The cells in some embodiments are co-administered with one ormore additional therapeutic agents or in connection with anothertherapeutic intervention, either simultaneously or sequentially in anyorder. In some contexts, the cells are co-administered with anothertherapy sufficiently close in time such that the cell populationsenhance the effect of one or more additional therapeutic agents, or viceversa. In some embodiments, the cells are administered prior to the oneor more additional therapeutic agents. In some embodiments, the cellsare administered after the one or more additional therapeutic agents. Insome embodiments, the one or more additional agents includes a cytokine,such as IL-2, for example, to enhance persistence. In some embodiments,the methods comprise administration of a chemotherapeutic agent.

In some embodiments, the method includes determining the biologicalactivity of the cellular engineering target, e.g., by any of a number ofknown methods. In some embodiments, one or more parameters utilized insuch a determination include specific binding of an engineered ornatural T cell or other immune cell to antigen, in vivo, e.g., byimaging, or ex vivo, e.g., by ELISA or flow cytometry. In certainembodiments, the method includes determining the ability of theengineered cells to destroy target cells using any suitable method knownin the art, such as cytotoxicity assays. In certain embodiments, themethod includes determining the biological activity of the cells byassaying expression and/or secretion of one or more cytokines, such asCD 107a, IFNy, IL-2, and TNF. In some embodiments, the method includesdetermining the biological activity by assessing clinical outcome, suchas reduction in tumor burden or load.

In some embodiments, the method includes providing a specific dosageregimen that includes a lymphodepletion step prior to the administrationof the modified T cells. In an exemplary embodiment, the lymphodepletionstep includes administrating cyclophosphamide and/or fludarabine.

In some embodiments, the administrating of lymphodepletion includesadministrating cyclophosphamide at a dose of between about 200mg/m.sup.2/day and about 2000 mg/m.sup.2/day (e.g., 200 mg/m.sup.2/day,300 mg/m.sup.2/day, or 500 mg/m.sup.2/day). In an exemplary embodiment,the dose of cyclophosphamide is about 300 mg/m.sup.2/day. In someembodiments, the lymphodepletion step includes administration offludarabine at a dose of between about 20 mg/m.sup.2/day and about 900mg/m.sup.2/day (e.g., 20 mg/m.sup.2/day, 25 mg/m.sup.2/day, 30mg/m.sup.2/day, or 60 mg/m.sup.2/day). In an exemplary embodiment, thedose of fludarabine is about 30 mg/m.sup.2/day.

In some embodiment, the administrating of lymphodepletion includesadministrating cyclophosphamide at a dose of between about 200mg/m.sup.2/day and about 2000 mg/m.sup.2/day (e.g., 200 mg/m.sup.2/day,300 mg/m.sup.2/day, or 500 mg/m.sup.2/day), and fludarabine at a dose ofbetween about 20 mg/m.sup.2/day and about 900 mg/m.sup.2/day (e.g., 20mg/m.sup.2/day, 25 mg/m.sup.2/day, 30 mg/m.sup.2/day, or 60mg/m.sup.2/day). In an exemplary embodiment, the administrating oflymphodepletion includes administrating cyclophosphamide at a dose ofabout 300 mg/m.sup.2/day, and fludarabine at a dose of about 30mg/m.sup.2/day.

In an exemplary embodiment, a subject has a diagnosis forcastrate-resistant prostate cancer, the method includes administratinglymphodepleting chemotherapy prior to administrating of the modified Tcellular engineering target. In an exemplary embodiment, for a subjecthaving castrate-resistant prostate cancer, the subject receiveslymphodepleting chemotherapy including at or about 500 mg/m.sup.2 to ator about 1 g/m.sup.2 of cyclophosphamide by intravenous infusion. In anexemplary embodiment, for a subject having castrate-resistant prostatecancer, the subject receives lymphodepleting chemotherapy including ator about 500 mg/m.sup.2 to at or about 1 g/m.sup.2 of cyclophosphamideby intravenous infusion about 3 days (.+−0.1 day) prior toadministration of the modified T cells. In an exemplary embodiment, fora subject having castrate-resistant prostate cancer, the subjectreceives lymphodepleting chemotherapy including at or about 500mg/m.sup.2 to at or about 1 g/m.sup.2 of cyclophosphamide by intravenousinfusion up to 4 days prior to administration of the modified T cells.In an exemplary embodiment, for a subject having castrate-resistantprostate cancer, the subject receives lymphodepleting chemotherapyincluding at or about 500 mg/m.sup.2 to at or about 1 g/m.sup.2 ofcyclophosphamide by intravenous infusion 4 days prior to administrationof the modified T cells. In an exemplary embodiment, for a subjecthaving castrate-resistant prostate cancer, the subject receiveslymphodepleting chemotherapy including at or about 500 mg/m.sup.2 to ator about 1 g/m.sup.2 of cyclophosphamide by intravenous infusion 3 daysprior to administration of the modified T cells. In an exemplaryembodiment, for a subject having castrate-resistant prostate cancer, thesubject receives lymphodepleting chemotherapy including at or about 500mg/m.sup.2 to at or about 1 g/m.sup.2 of cyclophosphamide by intravenousinfusion 2 days prior to administration of the modified T cells.

In an exemplary embodiment, the method includes, a subject havingcastrate-resistant prostate cancer, administrating lymphodepletingchemotherapy including 300 mg/m.sup.2 of cyclophosphamide by intravenousinfusion 3 days prior to administrating the modified T cells. In anexemplary embodiment, for a subject having castrate-resistant prostatecancer, the method includes administrating lymphodepleting chemotherapyincluding 300 mg/m.sup.2 of cyclophosphamide by intravenous infusion for3 days prior to administrating the modified T cells.

In an exemplary embodiment, for a subject having castrate-resistantprostate cancer, the method includes administrating lymphodepletingchemotherapy including fludarabine at a dose of between about 20mg/m.sup.2/day and about 900 mg/m.sup.2/day (e.g., 20 mg/m.sup.2/day, 25mg/m.sup.2/day, 30 mg/m.sup.2/day, or 60 mg/m.sup.2/day). In anexemplary embodiment, for a subject having castrate-resistant prostatecancer, the method includes administrating lymphodepleting chemotherapyincluding fludarabine at a dose of 30 mg/m.sup.2 for 3 days.

In an exemplary embodiment, for a subject having castrate-resistantprostate cancer, the method includes administrating lymphodepletingchemotherapy including cyclophosphamide at a dose of between about 200mg/m.sup.2/day and about 2000 mg/m.sup.2/day (e.g., 200 mg/m.sup.2/day,300 mg/m.sup.2/day, or 500 mg/m.sup.2/day), and fludarabine at a dose ofbetween about 20 mg/m.sup.2/day and about 900 mg/m.sup.2/day (e.g., 20mg/m.sup.2/day, 25 mg/m.sup.2/day, 30 mg/m.sup.2/day, or 60mg/m.sup.2/day). In an exemplary embodiment, for a subject havingcastrate-resistant prostate cancer, the method includes administratinglymphodepleting chemotherapy including cyclophosphamide at a dose ofabout 300 mg/m.sup.2/day, and fludarabine at a dose of 30 mg/m.sup.2 for3 days.

It is known in the art that one of the adverse effects followinginfusion of CAR T cells is the onset of immune activation, known ascytokine release syndrome (CRS). CRS is immune activation resulting inelevated inflammatory cytokines. Clinical and laboratory measures rangefrom mild CRS (constitutional symptoms and/or grade-2 organ toxicity) tosevere CRS (sCRS; grade.gtoreq.3 organ toxicity, aggressive clinicalintervention, and/or potentially life threatening). Clinical featuresinclude high fever, malaise, fatigue, myalgia, nausea, anorexia,tachycardia/hypotension, capillary leak, cardiac dysfunction, renalimpairment, hepatic failure, and disseminated intravascular coagulation.Dramatic elevations of cytokines including interferon-gamma, granulocytemacrophage colony-stimulating factor, IL-10, and IL-6 have been shownfollowing CAR T-cell infusion. The presence of CRS generally correlateswith expansion and progressive immune activation of adoptivelytransferred cells. It has been demonstrated that the degree of CRSseverity is dictated by disease burden at the time of infusion aspatients with high tumor burden experience a more sCRS.

Accordingly, the present disclosure provides for, following thediagnosis of CRS, appropriate CRS management strategies that mitigateone or more physiological symptoms of uncontrolled inflammation withoutdampening the antitumor efficacy of the cellular engineering target(e.g., CAR T cells). CRS management strategies are known in the art. Forexample, in some embodiments, the method includes administratingsystemic corticosteroids to rapidly reverse symptoms of sCRS (e.g.,grade 3 CRS) without compromising initial antitumor response.

In some embodiments, the method includes administrating an anti-IL-6Rantibody. An example of an anti-IL-6R antibody is the Food and DrugAdministration-approved monoclonal antibody tocilizumab, also known asatlizumab (marketed as Actemra, or RoActemra). Tocilizumab is ahumanized monoclonal antibody against the interleukin-6 receptor(IL-6R). Administrating of tocilizumab has demonstrated near-immediatereversal of CRS.

In some embodiments, the method includes selecting and treating asubject having failed at least one prior course of standard of cancertherapy. For example, a suitable subject may have had a confirmeddiagnosis of relapsed prostate cancer. In some embodiments, the methodincludes selecting and treating a subject having had at least one priorcourse of standard of cancer therapy. For example, a suitable subjectmay have had prior therapy with at least one standard 17.alpha. lyaseinhibitor or second-generation anti-androgen therapy for the treatmentof metastatic castrate resistant prostate cancer.

In an exemplary embodiment, a suitable subject is a subject havingmetastatic castrate resistant prostate cancer. In an exemplaryembodiment, a suitable subject is a subject having metastatic castrateresistant prostate cancer having .gtoreq.10% tumor cells expressing PSMAas determined by immunohistochemistry analysis on fresh tissue.

In some embodiments, a suitable subject is a subject that hasradiographic evidence of osseous metastatic disease and/or quantifiable,non-osseous metastatic disease (nodal or visceral).

In some embodiments, a suitable subject includes an ECOG performancestatus of 0-1.

In some embodiments, a suitable subject exhibits adequate organfunction, as defined by: serum creatinine.ltoreq.1.5 mg/dl or creatinineclearance.gtoreq.60 cc/min; and/or serum total bilirubin<1.5.times.ULN;serum ALT/AST<2.times.ULN.

In some embodiments, a suitable subject exhibits adequate hematologicreserve as defined by: Hgb>10 g/dl; PLT>100 k/ul; and/or ANC>1.5 k/ul.

In some embodiments, a suitable subject is not transfusion dependent.

In some embodiments, a suitable subject is a subject that has evidenceof progressive castrate resistant prostate adenocarcinoma, as definedby: castrate levels of testosterone (<50 ng/ml) with or without the useof androgen deprivation therapy; and/or evidence of one of the followingmeasures of progressive disease: soft tissue progression by RECIST 1.1criteria, osseous disease progression with 2 or more new lesions on bonescan (as per PCWG2 criteria), increase in serum PSA of at least 25% andan absolute increase of 2 ng/ml or more from nadir (as per PCWG2criteria).

In some embodiments, a suitable subject has had previous treatment withat least one second-generation androgen signaling inhibitor. In someembodiments, a suitable subject has had previous treatment withabiraterone. In some embodiments, a suitable subject has had previoustreatment with enzalutamide.

In some embodiments, a suitable subject includes .gtoreq.10% tumor cellsexpressing PSMA by immunohistochemistry (IHC) on a metastatic tissuebiopsy.

In some embodiments, a suitable subject includes radiographic evidencefor metastatic disease (osseous or nodal/visceral).

In some embodiments, a suitable subject includes .ltoreq.4 lines oftherapy for metastatic CRPC.

Additional details and information regarding the manufacture of cellularengineering targets can be found at U.S. Pat. No. 10,780,120, entitled“Prostate-specific membrane antigen cars and methods of use thereof,”filed Mar. 5, 2019; U.S. Pat. No. 10,839,945, entitled “Cell processingmethod,” filed Jul. 6, 2015; U.S. Pat. No. 10,428,351, entitled “Methodsfor transduction and cell processing,” filed Nov. 4, 2015; U.S. Pat. No.10,877,055, entitled “Parallel cell processing method and facility,”filed Jan. 11, 2019, each of which is hereby incorporated by referencein its entirety for all purposes.

Referring to FIG. 2, the computer system 100 is configured to store aninstrument library 106 describing a plurality of instruments 52 of arespective modular biological foundry system, 200.

In some embodiments, the server 200 receives the data elementswirelessly through radio-frequency (RF) signals. In some embodiments,such signals are in accordance with an 802.11 (Wi-Fi), Bluetooth, orZigBee standard.

In some embodiments, the computer system 100 is not proximate to thebiological foundry 200 and/or does not have wireless capabilities orsuch wireless capabilities are not used for the purpose of providinginstructions to the instruments 300 of the biological foundry 200. Insuch embodiments, a communication network 106 is utilized to communicatean update for executing a respective instance of a correspondingcompiled workflow from the service to the biological foundry. In someembodiments, the communication network 106 is utilized to communicate aresult of a manufacture of a respective cellular engineering targetproduced at the biological foundry 200 to the computer system 100.

Examples of communication networks 106 include, but are not limited to,the World Wide Web (WWW), an intranet and/or a wireless network, such asa cellular telephone network, a wireless local area network (LAN) and/ora metropolitan area network (MAN), and other devices by wirelesscommunication. The wireless communication optionally uses any of aplurality of communications standards, protocols and technologies,including but not limited to Global System for Mobile Communications(GSM), Enhanced Data GSM Environment (EDGE), high-speed downlink packetaccess (HSDPA), high-speed uplink packet access (HSUPA), Evolution,Data-Only (EV-DO), HSPA, HSPA+, Dual-Cell HSPA (DC-HSPDA), long termevolution (LTE), near field communication (NFC), wideband code divisionmultiple access (W-CDMA), code division multiple access (CDMA), timedivision multiple access (TDMA), Bluetooth, Wireless Fidelity (Wi-Fi)(e.g., IEEE 802.11a, IEEE 802.11ac, IEEE 802.11ax, IEEE 802.11b, IEEE802.11g and/or IEEE 802.11n), voice over Internet Protocol (VoIP),Wi-MAX, a protocol for e-mail (e.g., Internet message access protocol(IMAP) and/or post office protocol (POP)), instant messaging (e.g.,extensible messaging and presence protocol (XMPP), Session InitiationProtocol for Instant Messaging and Presence Leveraging Extensions(SIMPLE), Instant Messaging and Presence Service (IMPS)), and/or ShortMessage Service (SMS), or any other suitable communication protocol,including communication protocols not yet developed as of the filingdate of the present disclosure.

Of course, other topologies of the system 10 other than the one depictedin FIG. 1 are possible. For instance, in some embodiments, rather thanrelying on a communications network 106, the computer system 100wirelessly transmits information directly to the biological foundry 200.Further, in some embodiments, the computer system 100 constitutes aportable electronic device, a server computer, or in fact constituteseveral computers that are linked together in a network or areinstantiated as one or virtual machines and/or containers in acloud-computing context. As such, the exemplary topology shown in FIG. 1merely serves to describe the features of an embodiment of the presentdisclosure in a manner that will be readily understood to one of skillin the art.

Additional details and information regarding workflows at a biologicalfoundry system can be found at International Patent Application no.:PCT/US2021/018927, entitled “Systems and Methods for FacilitatingModular and Parallelized Manufacturing at a Biological Foundry,” filedFeb. 19, 2021, which is hereby incorporated by reference in itsentirety.

Accordingly, one aspect of the present disclosure is directed toproviding systems, methods, and apparatuses that facilitates providing amodular biological foundry system. The modular biological foundry systemincludes a controller and a communications interface that is inelectrical communication with the controller. Moreover, the modularbiological foundry system includes a plurality of peripheral devices,which in turn includes an articulated handling robot and a power supply.The articulated handling robot is configured to move a cell therapycartridge between at least a first biological foundry instrument and asecond biological foundry instrument in a plurality of biologicalfoundry instruments configured to produce a portion of the one or morecellular engineering targets. The modular system includes a framesurrounding the articulated handling robot. The frame includes at leasttwo modules in a plurality of modules. A first module in the at leasttwo modules is configured to accommodate a respective biological foundryinstrument in the plurality of biological foundry instructions.Moreover, each module in the plurality of modules includes a pluralityof elongated members. Each module in the plurality of modules furtherincludes a first plurality of coupling mechanisms for coupling at leasttwo elongated members in the plurality of elongated members.Additionally, each module in the plurality of modules includes a secondplurality of coupling mechanisms for removably coupling a respectiveelongated member in the plurality of elongated members with the frame.Furthermore, each module in the plurality of modules includes aplurality of walls engaged with and supported by the at least twoelongated members in the plurality of elongated members. Accordingly,the plurality of walls forms an internal volume of a respective modulesealed from an environment, such that a modular biological foundrysystem is provided.

In some embodiments, the modular biological foundry system furtherincludes a transport path coupled to the articulated handling robot andin electrical communication with the communications interface. Thetransport path extends from a first end portion of a first wall in theplurality of walls to a second end portion of a second wall in theplurality of walls.

Turning to FIG. 3 through FIG. 6 with the foregoing in mind, considerthat a manufacturing process for a cellular engineering target isdivided into a plurality of steps. In each step, a differentmanufacturing task or sub-process is carried out. For instance, in someembodiments, the different manufacturing task includes incubation ofcell culture, isolation of one or more types of cells, viraltransduction, freezing, thawing, etc. Accordingly, the presentdisclosure provides a modular clean room biological foundry system thatutilizes a separate module (e.g., module 208-1 of FIG. 3) to conducteach step of the manufacturing process.

Each module 208 includes an instrument (e.g., instrument 300 of FIG. 1,instrument 300 of FIG. 7, instrument 300-2 of Figure of FIG. 15, etc.)that performs a particular step (or set of steps) in a cellularengineering target manufacturing process. Accordingly, in someembodiments, each module includes both the instrument that carries outthe task, and/or one or more support instruments for that task. Supportinstruments includes air filtering systems (e.g., air filtering system220 of FIG. 4), electronic systems (e.g., electronic systems 212 of FIG.3), processors (e.g., CPU 202 of FIG. 2), cloud connectivity devices(e.g., communications network 106 of FIG. 1), and one or more reservoirsof raw material or waste material of the manufacturing process. A modulehas a well-defined shape formed through elongated members (e.g.,elongated members 216 of FIG. 6), which allows for the module to changepositions within the modular biological foundry without requiringmodification to the frame of the modular biological foundry system.Accordingly, a plurality of modules, together, realize the manufacturingprocess for producing a plurality of cellular engineering targets.

The modular clean room biological foundry system 200 includes a frame(e.g., frame 206 of FIG. 3, frame 206 of FIG. 5, etc.), such that eachmodule 208 removably couples with the frame 206. In some embodiments,the frame is formed from a plurality of elongate members (e.g.,elongated members 216 of FIG. 3), such that each elongated member formsan edge of the frame and couples to a different elongated member. Insome embodiments, the frame is located in proximity of a roboticmaterial transfer system (e.g., articulated handling robot 202 of FIG.3, transport path 204 of FIG. 4, etc.) that is able to move a portion ofa cellular engineering target, such as a rigid cartridge accommodatingthe cellular engineering target (e.g., rigid cartridge 1100 of FIG. 14),among the different modules of the modular clean room biological foundrysystem. For example, when manufacturing a new cellular engineeringtarget, the robotic material transfer system brings a plurality ofinitial reagents to a first module 208-1 that is necessary for aninitial process of the manufacture of the new cellular engineeringtarget. Once the first module has performed its task, the materialtransfer system moves the cellular manufacturing target to the secondmodule required by the manufacture process. In some embodiments, thesesteps are repeated (e.g., moving a portion of a cellular engineeringtarget from a first module to a second module of the frame) for eachsuccessive task in the manufacture, until the manufacture of thecellular engineering target is complete.

Since this biological foundry system is modular, additional modules 2087can be added to frame 206 if needed. For example, in some embodiments,additional modules added to the increases production capacity at modularbiological foundry system 200. Should a new manufacturing task berequired to realize a new or modified cellular engineering target, themodules of the modular biological foundry system allow for adding and/orremoving modules in order to facilitating the manufacture of the new ormodified cellular engineering targets. Additionally, this modulararchitecture enables parallel production. Additionally, in someembodiments, unique engineering targets in a plurality of cellularengineering targets can be realized by the modular biological foundrysystem at the same time, if there are enough individual modules 208 tocarry out all the required tasks simultaneously. In some embodiments,one or more buffers is added to the modular biological foundry system,in order to increase the capacity of the modular biological foundrysystem to manufacture a plurality of cellular engineering targets at thesame time.

In some embodiments, modules 208 are placed in frame 206 that isequipped with a plurality of coupling mechanism (e.g., couplingmechanism 216 of FIG. 4) that facilitating holding a respective modulein place within the frame. Accordingly, in some embodiments, the framefacilitates accommodating a plurality of modules on a plurality of rowsand/or columns of modules. For example, referring briefly to FIG. 5, insome embodiments, two or three modules 208 is stacked vertically and/orhorizontally into a plurality of rows and/or columns and supported inplace by the frame. This configuration would respectively support two orthree times more modules than a configuration with a single columnand/or row. In some embodiments, the frame allows accommodating aplurality of module side by side, either along straight lines (e.g.modules are side by side in a row, such as forming a substantiallyrectangular shape) or along a curved line (e.g. modules are disposed ina substantially circle shape around articulated handling robot 202and/or transport path 204). In this way, the frame makes it easy toaccommodate modules within the frame, add new modules to the frame, andremovable couple modules that are no longer needed from the frame, thusproviding a modular biological foundry system. Because of the frame, thebiological foundry system can be continuously adapted and adjusted bymodify a first subset of modules in without affecting a second subset ofmodules that are not being modified.

Accordingly, in some embodiments, the modular biological foundry systemincludes the modules, the frame, and the robotic material transfersystem (e.g., articulated handling robot, transport path). Moreover, insome embodiments, the biological foundry system includes one or moresupport instruments (e.g. air filtering and discharge systems 220,electronics 212, power supply, controller, communications interface,etc.) that is included in the modules and/or the frame, or physicallyattached to them.

Each module 208 includes an external structure that includes a pluralityof elongated members (e.g., elongate members 216 of FIG. 6).Collectively, the plurality of elongated members couple at respectiveend portions to form an interior of the module that accommodates acorresponding instrument. In this way, the elongated members form theedges of each module 208. The instrument is utilized for carrying out astep (or set of steps) in the manufacture of a cellular engineeringtarget, such as a soft body bioreactor (e.g., soft body bioreactor 1200of FIG. 13). In some embodiments, this soft body bioreactor is placedinside of a respective module. The respective module will then provide acontrolled environment for the soft body bioreactor to operate in, andprovide electrical power one or more mechanisms (e.g., sensors) thatfacilitates obtaining data elements as inputs and/or outputs for thesoft body bioreactor. This way, both custom-built and commerciallyavailable instruments can be integrated into a respective module of themodular biological foundry system.

In some embodiments, except for a respective instrument 300 accommodatedby each module (e.g., instrument that performs a manufacture task), eachmodule shares the same external structure, such that the frame canaccommodate each respective module in a plurality of modules at aposition of the frame. As used here, an external structure of arespective module is a “shell” of the respective module. Accordingly,the shell provides the respective module with a base platform (e.g.,base 230 of FIG. 3), on which an instrument 300 is supported. Inaddition to providing a base for the respective module, the shell canalso provide a plurality support functions that support the biologicalfoundry system and/or instrument of the respective module in themanufacture of cellular engineering targets. These support functionsinclude air filtering systems, air removal systems, power supply,controller(s), computing devices (e.g., computer system 100 of FIG. 2),cloud connectivity devices (e.g., communications network 106 of FIG. 1),or a combination thereof. However, the present disclosure is not limitedthereto.

In some embodiments, the shell of the respective module 208 is selectedfrom a group consisting of at least two types of shells, at least threetypes of shells, at least four types of shells, at least five types ofshells, at least ten types of shells, or a combination thereof.

In some embodiments, a shell of a respective module includes a baseplatform and a plurality of elongated members (e.g., forming a hollowbox shell). Each end portion of an elongated member 216 is coupled toanother end member of a second elongated member, to form the edges ofthe module and/or frame. Collective, the elongated members and/or baseform the frame. In such embodiments, the shell of the respective moduleincludes a substantially flat base on which an instrument is supportedand elongated members that surrounds the instrument on all sides. Insome embodiments, a front side of the instrument is exposed, such asmodule 208-2 of FIG. 3 exposing instrument 300-4 through front sidefacing articulated robot 202). In such embodiments, the shell of therespective module provides an interior that protects the instrument fromall sides except for the front, where it presents an access opening forthe robotic material transfer system. This shell type also allows therespective module to be precisely mounted on the frame of the modularbiological foundry system. For instance, in some embodiments, the baseand/or the elongated members of the respective module is removablycoupled to the frame with a coupling mechanism, such as one or moreclamps, one or more bolts, or the like.

In some embodiments, the shell includes an electronics enclosure (e.g.,electronic cabinet 212 of FIG. 5). The electronics enclosure includes aninterior portion of the respective module that is separated from therest of the respective module (e.g., by base platform 230). Theelectronic enclosure provides the interior portion for accommodating aplurality of electronic associated with the instrument and/or biologicalfoundry system, such as controller(s), power supply, and communicationsnetwork interface (e.g., network interface 284 of FIG. 2). In someembodiments, the electronics enclosure is typically positioned at abottom end portion of the module below the base platform on which aninstrument is accommodated. From this, wires from/to the instrument canbe routed through an aperture of the base platform (e.g., with sealedjoints), and then reach electronics of the electronics solution.Accordingly, this configuration provides superior ease of assembly andtesting, while also protecting electronics from fluids or reagents thatcould be spilled as a result of a manufacturing and/or cleaning processconducted inside and/or surrounding a module. However, one of skill inthe art will appreciate that the present disclosure is not so limited.

For instance, in some embodiments, the shell of the respective module208 includes an air filtering system 220. The air filtering systemprovides the respective module with a controlled flow of clean air, and,optionally, removes (e.g., exhaust) waste air from the respective modulewhen needed. In some embodiments, the air filtering system is disposedin an upper end portion of the shell of the respective module. This way,the air filtering system can generate a top-down flow of clean air abovean instrument in the respective module, such as a laminar flow of fluidthrough the modular biological foundry system. In alternativeembodiments, the air filtering system is disposed in a lateral portionof the respective module, such that the air filtering system provides ahorizontal flow of fluid (e.g., horizontal laminar flow throughbiological foundry system). In such embodiments, the fluid (e.g., air)that is pushed and/or drawn by the air filtering system exits therespective module through an open side portion of the respective module,such as the front side of the respective module, which is the sameopening through which the robotic material transfer system accesses themodule. However, the present disclosure is not limited thereto. In someembodiments, the base platform and/or a side portion of the respectivemodule includes a second air filtering system that facilitates wasteremoval. In some embodiments, the air filtering systems includes one ormore perforated side panels coupled to the elongated members of themodule, filtering membranes, filtering ducts, louver vents, and thelike. One of skill in the art will appreciate that the presentdisclosure is not so limited. This way, in some embodiments, fluid thatis pushed in from an upper end portion of the respective module by theair filtering system is then removed at a bottom end portion of therespective module by the second air filtering system for waste removal.Accordingly, the air filtering system provides a module with a cleanroom environment, because the fluid inside of the module is filtered andcontrolled (e.g., by controller 288 of FIG. 2).

As described supra, modules 208 are held in place by frame 206. Astructure of the frame makes it simple and quick removably couplemodules with the frame. For instance, in some embodiments, modules areslid into the frame with one or more guiding mechanism, such as one ormore jigs, one or more linear guides, one or more stoppers, one or morerails, one or more bearings, or the like.

In some embodiments, each respective module includes a sealing mechanism218, such as a gasket, that creates a seal between an interior of arespective module and the modular biological foundry system. In someembodiments, once a respective module 208 has been disposed at aposition within frame 206, the module is then pressed onto a gasket(e.g., gasket 218 of FIG. 6) that is located in front of the module(e.g., open front side of module 208 of FIG. 6). In some embodiments,gasket 218 surrounds a perimeter of a side of a module, such ascontinuously on the whole perimeter of the front face of the module. Insome embodiments, the side of the module including the gasket is thesame side where there is an opening that allows the robotic materialtransfer system access to the interior of the module. In suchembodiments, the gasket is positioned around this opening, so that whenthe module is disposed in the frame, the module is pressed against thegasket and the frame to create a clean-room grade seal between themodule and the frame. In other words, if the frame of the modularbiological foundry system is a clean room environment, adding a moduleto the frame and ensuring that the gasket is engaged will not compromisethe environmental controls on the clean room of a central space of themodular clean room biological foundry system, such as space 222 of FIG.4 that accommodates an articulated handling robot 202 and transport path204. Accordingly, the module that is added to the frame will constitutean addition to the clean room space 222 of the modular biologicalfoundry system without risking contamination to the clean room spaceand/or other modules of the frame.

In some embodiments, coupling mechanism 216 apply a force to the module208 against the frame 206, and therefore the gasket 218, connecting themodule to the frame in a clean-room-compatible manner. These couplingmechanisms, exerting a force on a rear portion (or on other regions) ofthe module and pushing the module towards the gasket, include one ormore clamps (mechanism 216 of FIG. 4), one or more screws, one or morelevers, one or more detent mechanisms, or a combination thereof.

In some embodiments, once module 208 has been connected to frame 206with sealing mechanism 218, the module can also be removed from theframe without destroying the clean room conditions of the modularbiological foundry system. The disposal and removal processes for eachmodule is equally simple and rapid. The module reduces the engineeringand retooling costs associated with modifying the configuration of themodular biological foundry system. More modules can be added to theframe when desired or needed. For instance, modules that are no longeruseful in the manufacture of a cellular engineering target can beremoved from the frame to make room for other modules. If a newmanufacturing task needs to be performed, an appropriate new module canbe added to the frame. All these operations are executed quickly,without engineering or retooling costs, given the uniformity of themodules and the frame. The result is a flexible manufacturing system(i.e., the modular biological foundry system) that can adapt todifferent processes and different production scales as needed. Thislevel of flexibility and scalability is impossible with traditionalserial production systems, which are “locked in” in the initialconfiguration.

In some embodiments, each module 208 is separated from frame 206 ofmodular biological foundry system 200 by a second sealing mechanism(e.g., sealing mechanism 214 of FIG. 5). Sealing off a module from themodular biological foundry system is essential when a module is removedfrom modular biological foundry system. For example, if a first modulemalfunctions—or if it must be replaced with a second module differentthan the first module—it is important to remove the first module withoutaffecting the regular clean-room operational conditions of the modularbiological foundry system, such as disturbing a third module and/or arigid cartridge. This is achieved with second sealing mechanism 214 thatis activated before removing the module from the frame. The secondsealing mechanism creates a physical barrier between the module and themodular biological foundry system, such as space 222 of FIG. 4. By doingso, the second sealing mechanism ensures that the module to be removedfrom the frame does not affect the clean room conditions inside themodular biological foundry system.

Second sealing mechanism 214 can be implemented with a variety ofembodiments, including a multi-segmented sliding doors mechanism (secondsealing mechanism 214 of FIG. 4). This multi-segmented sliding doorsecond sealing mechanism can be easily operated by the robotic materialtransfer system (e.g. articulated handling robot 202), by sliding anouter segment from one end portion of the module to a second end portionof the module, and opening or closing the second sealing mechanism.Since the sliding door second sealing mechanism is composed of multiplesegments, it occupies a limited space when it is folded. The moresegments the sliding door has, the less space it occupies in the foldedposition. However, one of skill in the art will appreciate that thepresent disclosure is not limited thereto.

By way of example, in some embodiments, second sealing mechanism 214includes a horizontally oriented sliding door mechanism. For instance,referring briefly to FIG. 5, third module 208-3 includes third secondsealing mechanism 214-3 with four horizontal segments. The advantage ofthis second sealing mechanism is that the robotic material transfersystem that operates the second sealing mechanism does not have to workagainst gravity to open or close the second sealing mechanism.

In some embodiments, second sealing mechanism 214 includes mechanicalmechanism 224 that is disposed on an exterior portion of the secondsliding mechanism, such as a round knob, a cylindrical knob, ahemispherical knob, a crank, or the like. For instance, referringbriefly to FIG. 5, second module 208-2 includes first second sealingmechanism 214-1 that is a door with first mechanical mechanism 224-1that is a handle protruding outwardly from an exterior surface of thefirst second sealing mechanism). This includes mechanical mechanismenables the robotic material transfer system to easily operate thesecond sealing mechanism without exerting unwanted torques. For example,the robotic material transfer system could feature a manipulator shapedlike a hollow tube, such as a hollow tube including a lead-in edgeincluding a chamfer on the edge of an inner hole of the hollow tube.This manipulator can be easily positioned around the circular knob,enabling the robotic material transfer system to open and close thesecond sealing mechanism and access the interior of the module.

In alternative embodiments, second sealing mechanism 214 include asliding door mechanism that opens and closes by sliding in a verticaldirection. For instance, referring briefly to FIG. 5, fourth module208-4 includes third second sealing mechanism 214-3 that includes fourvertical segments. This vertical second sealing mechanism isadvantageous if, in its folded position, it substantially overlaps witha region of the module other than an opening used to access the interiorof the module by the robotic material transfer system, such aselectronics enclosure 212 and/or air filtering mechanism 220. This way,the opening in the front of the module would be larger when the slidingmechanism is folded.

In some embodiments, second sealing mechanism 214 includes a rigid panelthat removably couples to frame 206 and/or module 208. For instance, insome embodiments, the second sealing mechanism includes magnetic locksthat couple to the frame of modular biological foundry system 200, suchas in front of an opening of the module that is accessed by roboticmaterial transfer system 202. Accordingly, the robotic material transfersystem (e.g. an articulated handling robot) can handling the secondsealing mechanism that is a rigid panel and separate the interior of themodule from the modular biological foundry system with the rigid panelwhen it is necessary to separate a respective module from the modularbiological foundry system. In some embodiments, the second sealingmechanisms sealing is stored inside the modular biological foundrysystem, so that the robotic material transfer system can rapidly pickone second sealing mechanism and use the second sealing mechanisms toseal a respective module whenever such sealing is needed.

In some embodiments, the second sealing mechanisms 214 includes one ormore rolling door mechanisms, one or more pivoting door mechanisms, oneor more multi-segmented pivoting door mechanisms, sliding panelmechanisms that can slide over one or more modules, and the like.

In some embodiments, a rear portion of module 208 there is an accessmechanism that allows an operator to access the module for maintenanceand to perform manual operations, such as installing instrument 300within an interior of the module. For instance, in some embodiments, theaccess mechanism includes a door. In some embodiments, the rear portionof the module includes one or more spaces, or apertures, to insert orremove rigid cartridges 1100.

In some embodiments, rigid cartridge 1100 is utilized to store amaterial utilized in the manufacture of a cellular engineering target,such as cell culture media, reagents, pharmaceutical ingredients, etc.).In some embodiments, the cartridge is initially an empty container thatwill then accommodate a material, such as waste material produced as aninstrument accommodated by the module performs a manufacturing task.Accordingly, the presence of apertures that facilitate accommodatingcartridges on the rear end portion of the module allows a user to insertnew cartridges and remove old cartridges from the apertures withoutaffecting the sterility or the clean-room conditions inside the moduleand the rest of the modular biological foundry system. In someembodiments, the apertures are the rear end portion of the moduleinclude physical barrier that separates an external environment (e.g.,atmosphere) from the interior of the module. In some embodiments, therear of the module includes a plurality of ports and/or a plurality ofvalves that is accommodated by the apertures, which allows the modularbiological foundry system to connect an interior of the cartridge withthe instrument in the module, such as once the cartridge has beenremovably coupled with a respective aperture at the rear end portion ofthe module. These ports and/or valves enable a sterile connectionbetween the cartridge and the instrument of the module, such that acartridge replacement process at a respective module is performedwithout any external fluids (e.g., atmospheric air) or contaminantsentering the module. A user outside of the modular biological foundrysystem can add and remove the cartridges, without affecting orcompromising the clean-room conditions inside of the module and themodular biological foundry system.

The modules are placed within frame 206. The frame is a rigid structurethat accommodates the modules 208. In some embodiments, the frameincludes a plurality of beams, a plurality of planar or substantiallyplanar surfaces, one or more sealing mechanisms (e.g., sealing mechanism218), one or more second sealing mechanisms 224, one or more conduitsfor wires and the like, one or more electronic systems accommodated byelectronics enclosure 212, one or more air filtering mechanisms 220, ora combination thereof. One of skill in the art will appreciate that anyother feature or sub-system that is necessary to support the operationof the modules within the modular biological foundry system is withinthe domain of the present disclosure. In this way, the frame keeps themodules in a fixed, pre-determined position within the modularbiological foundry system 200. In this way, each module and/orinstrument is associated with a fixed address (e.g., address ofinstrument 108 of FIG. 2, address of module 112 of FIG. 2, which is usedby the robotic material transfer system for spatial determinations whenmoving the cartridge. In some embodiments, the frame includes electricalpower and/or network interface connectivity for communicating withcomputer system 100 over communications network 106. In this way, theframe allows the modules to operate into a shared clean-roomenvironment, where the manufacturing process to produce the cellularengineering targets can be carried out in a sterile manner.

The frame can host multiple rows of modules 208 and multiple columns ofmodules 208. For instance, in some embodiments, the frame includes anarrangement of modules 208 in which the modules are placed in a straightline, in a rectangle, in a circle, or any other straight or curvilinearshape (e.g., an open shape, a closed shape, or a combination of bothopen and closed shapes).

In some embodiments, modular biological foundry system 200 includesmultiple frames 206 that are placed side by side, creating aisles orgroups of frames 206. In some embodiments, each aisle or group of frames206 includes a unique transport path and/or articulated handling robot.In other embodiments, the aisles or groups of frames 206 share one ormore transport paths and/or articulated handling robot. For instance, insuch embodiments, a single robot material transfer system can facilitateconducting the manufacture of cellular engineering targets are eachinstrument 300 of each module 208 of each aisle or groups of frames 206while moving one or more cartridges 1100 between each instrument 300 oreach module 208.

In some embodiments, besides accommodating modules 208, frame 206includes one or more solid side walls or panels that seal an interior ofthe modular biological foundry system from an environment. In this way,the solid side walls provide a self-contained clean room that iscompletely isolated from the outside environment. Each module 208interfaces with this clean room environment of the frame through anopening on a front end portion of a module. In some embodiments, theopen front end portion of the module is sealable, in case the moduleneeds to be removed, such as by the sealing mechanism 214, 224 describedsupra. Inside the frame, the whole environment is controlled (i.e. it'sa sterile clean room). And, inside the frame, there are only roboticsystems. No human operator is present within the modular biologicalfoundry system while the system is in operation. This ensures theefficiency of the system and prevents external sources of contamination(including human operators) from affecting the process.

In some embodiments, a lower end portion of frame 206 includes anenclosure that hosts the electronic systems providing power and networkconnectivity to all the modules hosted in the frame. In someembodiments, an upper end portion of the frame includes air filteringmechanism 220 (e.g., air filtering mechanism 220 of FIG. 4), whichensures that the environment provided by the modular biological foundrysystem where the robotic material transfer system operates (between thevarious modules of the frame) is clean-room grade and sterile. In someembodiments, in the bottom part of the vertical walls of the frame thereare discharge filters, which allow the air at the bottom of the modularbiological foundry system to exit—while the higher internal pressureprevents external air from infiltrating modular biological foundrysystem. The whole volume that is included inside the frame and themodules is completely closed off from the external environment. Thisallows the modular biological foundry system to operate as anindependent clean room within an external environment that might have alower level of control.

In some embodiments, at a lower end portion of frame 206, a sealingmechanism, such as a gasket, ensures that there is a seal between theelongated members of the frame that accommodate the modules and a base(e.g., transport path 204) that supports the robotic material transfersystem. In some embodiments, this sealing mechanism is a gasket (e.g.,gasket 218 of FIG. 6) between a lower end portion of the frame (whichoptionally includes the modules) and the base structure of the roboticmaterial transport system. This way, the frame and the robotic materialtransfer system can be designed as separate systems and assembled inplace. Additionally, the sealing mechanism at the lower end portion ofthe frame ensures that the lower end portion of the modular biologicalfoundry system is completely sealed off from an environment, such thathere is a continuous separation between the interior space 222 of themodular biological foundry system and the environment where the modularbiological foundry system is located.

In some embodiments, the modular biological foundry system is not onlyprotected from external air infiltrating it. It is also protected, atthe level of the production floor, from cleaning liquids or reagentsinfiltrating it. For example, when manufacturing operators mop theproduction floor, the gasketed structure of the bottom of the modularbiological foundry system prevents that splashes or drops of cleaningliquids infiltrate the internal clean-room environment.

In some embodiments, the frame makes it possible to isolate the internalclean-room environment of the modular biological foundry system from theexternal environment. This means that the modular biological foundrysystem can be placed (and operated) in lower-grade clean rooms, whileguaranteeing that the cells are manufactured in highly controlled,sterile conditions.

In some embodiments, the frame provides an area of about 100 square feet(sq. ft.), of about 150 sq. ft., about 160 sq. ft., about 175 sq. ft.,about 200 sq. ft., about 250 sq. ft., about 300 sq. ft., about 350 sq.ft., about 400 sq. ft., about 450 sq. ft., about 500 sq. ft., about 550sq. ft., about 600 sq. ft., about 650 sq. ft., about 700 sq. ft., about750 sq. ft., about 850 sq. ft., about 950 sq. ft., about 1000 sq. ft.,or about 1,500 sq. ft. In some embodiments, a height of the frame isabout 3 feet (ft), about 4 feet, about 5 feet, about 6 feet, about 7feet, about 8 feet, about 9 feet, about 10 feet, about 11 feet, or about12 feet. In some embodiments, a width of the frame is about 5 feet,about 6 feet, about 8 feet, about 10 feet, about 12 feet, about 14 feet,about 16 feet, about 18 feet, about 20 feet, about 24 feet, about 28feet, about 32 feet (e.g., 33 feet). However, the present disclosure isnot limited thereto.

Additionally, in some embodiments, creating an isolated production spaceinside the modular biological foundry system enables the use of a widerange of systems to enforce sterility. These systems can be employedbecause, within the modular biological foundry system, there are nohuman operators. Therefore, sterilization and cleaning systems thatwould be dangerous for human operators can be used inside of the modularbiological foundry system, while the frame makes sure that they areincluded in the controlled environment. This protects the manufacturingpersonnel that operate outside the modular biological foundry system,while ensuring superior sterility inside the modular biological foundrysystem.

In some embodiments, automatic systems to ensure the sterility of theclean-room environment inside the modular biological foundry systeminclude, but are not limited to, the use of: gases (like ozone or CO₂),ultraviolet light, chemical cleaning agents (sprayed or in liquid form),radiation, extreme temperatures (high or low), etc. Multiplesterilization techniques can also be applied at the same time, or atdifferent times during the same manufacturing process. In all cases, thefact that the frame isolates the inside of the modular biologicalfoundry system from the outside allows the system to resort to the mostappropriate sterilization techniques, without endangering the humanoperators.

A further advantage is that these cleaning methods can be executed by arobotic arm or by another automatic system within the modular biologicalfoundry system. This ensures that the cleaning/sterilization proceduresare executed in a repeatable, measurable, and validated manner.

In some embodiments, the manufacturing process of a cellularmanufacturing target is completed by moving each cellular manufacturingtarget t from one module to the next, so that each machine can performits task on the cellular manufacturing target. Note that, in the contexton individualized medicine, the size of a batch is one—meaning that eachbatch includes only one product, because each cellular manufacturingtarget is different from the others. This robotic manufacturingarchitecture is very efficient when managing small batches, i.e. batchescomposed of a few products. The limit case is that a batch includes asingle product. Batches that include a few identical products can alsobe managed easily, by “entraining” a set of products that aremanufactured one after the other by the same sequence of modules.

In all module and batch configurations, it is essential to have arobotic material transfer system that moves the products between thedifferent modules of the modular biological foundry system. The roboticmaterial transfer system accesses each module through an opening in thefront side of the module. A wide range of robotic material transfersystems can be used in modular biological foundry system, including butnot limited to: a robotic arm on a rail, a robotic gantry, a conveyorbelt system, a magnetic rail system, a wheeled robot moving on thefloor, a legged robot, an aerial robot, etc. In some embodiments, therobotic material transfer system includes a flexure gripping device(e.g., included as a hand of an articulated handling robot 202).Additional details and information regarding a flexure gripping devicecan be found at U.S. Pat. No. 10,773,392, entitled “Flexure GrippingDevice,” filed Mar. 7, 2019, which is hereby incorporated by referencein its entirety.

In some embodiments, a plurality of robotic material transfer systems isdisposed within modular biological foundry system 200. For example, insome embodiments, the robotic material transfer systems includes aconveyor belt (e.g., coupled to a side portion of frame 206) that can beused to quickly supply reagents and the, such as while an articulatedhandling robot performs operations within modules 208 that is lessfrequent or less predictable. In some embodiments, computer system 100allows for utilizing different robot material transfer systems forvarious payloads. As a non-limiting example, in some embodiments, afirst articulated handling robot 202-1 is utilized to transport smalland/or lighter items (e.g., rigid cartridge 1100 of FIG. 18C), whereas asecond articulated handling robot 202-4 is utilized to transport largerand/or heavier items. This flexibility in the robotic material transfersystem is particularly relevant for a manufacture of cellularengineering targets, where, in some embodiments, by adding a media tosustain a cellular growth, a batch of cellular engineering targets canstart from a few milliliters in volume and increase in volume up toseveral liters of liquid material. Additionally, in some embodiments, aplurality of robotic material transfer systems is combined to provideincreased throughput when manufacturing cellular engineering targets atthe modular biological foundry system. For example, in some embodiments,a first material transport system (e.g., a first articulated handlingrobot and/or first conveyor belt) and a second material transport system(e.g., a second articulated handling robot and/or second conveyor belt)provides more throughput for the modular biological foundry system thanjust using a single robotic material transfer system.

Since the modular biological foundry system is a completely closed offclean room, in some embodiments, the modular biological foundry systemincludes a gate mechanism to receive input material and to outputfinished products. In some embodiments, this gate system includesdedicated, automatic airlock. In such embodiments, the airlock of thegate is the only point of the modular biological foundry system whereinput materials can be inserted, and output products (finished cellularengineering targets or quality control samples) can be extracted.

In some embodiments, the airlock gate mechanism includes but not limitedto a double-door mechanism (e.g., a first door and a second door eachprovide access to an interior of the gate mechanism). In thisembodiment, a user can open an external door of the gate mechanism andplace an input product inside of the interior. In some embodiments, theexternal door is then locked via a locking mechanism, such as a hatch ora key. In some embodiments, each door includes a sealing mechanism, suchas a gasket 218, to ensure a clean room environment. However, thepresent disclosure is not limited thereto. In some embodiments, themodular biological foundry system determines that the external door islocked (e.g., via vision system, via light gate, via sensor, etc.,), adecontamination routine is performed that prepares the product (and theenvironment that surrounds the product in the airlock, such as theinterior of the gate mechanism) to be inserted into the clean-roomenvironment of the modular biological foundry system. In someembodiments, once the decontamination routine is completed, the modularbiological foundry system autonomously opens the internal door of theairlock, and the robotic material transfer system is then able toretrieve the input product. In some embodiments, as a final step of theoperation cycle, for the airlock gate mechanism, is represented byautomatically closing the internal door. In some embodiments, this cycleis repeated in the opposite order when there are cellular engineeringtargets that need to be extracted from the modular biological foundrysystem—like completed cellular engineering targets or samples forquality control. When cellular engineering targets (e.g., rigidcartridge including cellular engineering targets) are extracted from themodular biological foundry system, the decontamination cycle begins onlyafter the external door of the airlock gate mechanism has been openedand then closed. Accordingly, in some embodiments, the aforementioneddecontamination process follows contact with the external environment,since the internal environment of the modular biological foundry systemis already controlled and sterile by air filtering system 220 andsealing mechanism 214, 218, 224.

In some embodiments, since no human user is inside the airlock gatemechanism when both of the doors are locked, it is possible for themodular biological foundry system to use the same decontaminationprocess that can be applied inside of the modular biological foundrysystem. For instance, because the airlock gate mechanism is separatedfrom the external environment, these decontamination process do not posea danger to the human user, who are outside of the modular biologicalfoundry system and the airlock gate mechanisms when the modularbiological foundry system and the airlock gate mechanisms are inoperation.

In some embodiments, material can be inserted into the modularbiological foundry system or extracted out of the modular biologicalfoundry through the rigid cartridges that can be placed in the rear endportion of each module 208. However, in such embodiments, this approachbased on the cartridges is used to provide raw materials and to removewaste materials from a respective module. In this way, in suchembodiments, the rigid cartridges are supporting tasks and materialsthat might be needed by the modules as they perform the manufacturingtasks on the products. However, the only point of the system that allowsfor the passage of full products (either at the initial stage—beforestarting the process—or when they are completed) is the airlock system.The airlock system also allows for the passage of sampling containers orvials, which are used to extract samples for the products inside themodular biological foundry systems. Once extracted from modularbiological foundry system, these samples can then be separately analyzedby human operators within a laboratory.

The vials or containers (e.g., rigid cartridges) including productsamples can also be transferred to the external environment via aseparate, smaller, high-throughput airlock. This is possible because thevials including these samples are small, and their flow isunidirectional (they are always coming from the inside of the modularbiological foundry system and going outside). Lots of clean, sterile,unused sampling vials or containers can be supplied to the modularbiological foundry system through special cartridges that can beinserted in a module without compromising the clean-room qualificationof the modular biological foundry system.

Inside the airlock system, a docking station enables the human operatorsto place the input materials in a fixed and well-controlled position, sothat they can be easily and repeatably picked up by the robotic materialtransfer system. The docking station also enables the robotic materialtransfer system to deposit outgoing products and quality controlsamples, so that they can be retrieved by the human operators outside ofthe modular biological foundry system.

Cell manufacturing processes are often characterized by unbalancedproduction steps. In other words, some steps in the cell manufacturingprocess take a significantly longer amount of time than the others.Consequently, some of the machines that are involved in a cellmanufacturing process have cycle times that are longer than the cycletimes of the other machines. For example, bioreactors typically requiredays (or even weeks) to complete cellular growth, while centrifuges areused for process steps that typically take only a few hours. Thiscreates an unbalanced process and makes the use of production lines(e.g., serial production architectures) structurally inefficient forcellular manufacturing applications.

The modular structure of the modular biological foundry systemcompensates for this imbalance by offering the possibility to addmultiple “slow” modules in parallel. An example is a modular biologicalfoundry system including multiple (dozens, or even hundreds) bioreactormodules that work in parallel. This way, while an individual bioreactormodule remains slow (the underlying process task has not changed, andthe cells take the same time to grow), the overall cycle time of themodular biological foundry system is greatly reduced—proportionally tothe number of bioreactor modules that are working in parallel. Thepossibility to overcome production bottlenecks by adding multiplemodules in parallel is a significant advantage of the parallel structureof the modular biological foundry system. Only parallel systems canquickly address unbalanced production steps by increasing the number of“slow” machines. Note that this does not require any additionalengineering cost or retooling time—the additional modules are simplymounted in the frame of the modular biological foundry system (which hasbeen designed for this purpose), easily addressing the productionbottleneck.

This modular biological foundry system architecture can also be employedto create groups of modules that coordinate their operations. Forexample, within a cell manufacturing modular biological foundry system,it is possible to create a group of “fast” modules that are operated bya fast robotic arm (i.e. the products are transferred between them by arapid robotic material transfer system). This group of fast modules iscomposed of modules that, for example, can each perform theirmanufacturing task in less than two hours. In this example, the overallcycle time of the group of fast modules is no longer than 2 hours. Andthis group of fast modules includes a single unit for each type ofmodule. Since all the modules contained in this group are fast, it isnot necessary to duplicate them in order to increase their average cycletime.

The group of fast modules described in this example can be paired,within the same modular biological foundry system, with a group of“slow” modules. The slow modules (which are typically, but notexclusively, bioreactor modules) take much longer to complete theirtask. For example, a bioreactor might need 1 day to complete its taskwithin the cell manufacturing process. In this case, the systems andmethods of the present disclosure would need to include 12 bioreactormodules in the group of “slow” modules, so that their average cycle timeis reduced to the same 2 hours cycle time that characterizes the fastmodules. In this configuration, the group of fast modules (include onlya first module for each fast module type) continuously feeds new batchesto a large group of slow modules (including multiple modules for eachslow module type). Each time a slow module is empty (because it hascompleted its task), the fast modules feed it with a new batch. Thegroup of slow modules is operated by a larger, slower robotic arm (i.e.a robotic material transfer system with higher payload and lower speed).This material transfer system can be slower because it needs to deposita batch in each position, and then leave it there for a long time, whilethe slow task is being completed. And this material transfer system canalso be larger (i.e. with a higher payload) because, especially in thecase of bioreactors, the volume of the product increases with time.Cells need more media as they grow, so the final size of the batch (atthe end of the bioreactor step) can be of several liters (as opposed toless than a liter at the beginning of that manufacturing step).Therefore, the slow robotic arm managing the bioreactors might need ahigher payload than the fast robotic arm managing the fast modules.

The architecture of the modular biological foundry system also allowsfor more modules and more robotic arms to be added when needed. Byadding more modules and more robotic material transfer systems, it ispossible to increase the throughput of the modular biological foundrysystem, making the process more rapid and more efficient.

The modular structure of the modular biological foundry system (whereeach individual module is mounted to the frame and can be separated by asealing system) allows to take any module offline in case there is afailure maintenance is needed. In other words, if a module fails orneeds to be serviced, it can be taken offline by sealing the door on itsfront side and by removing it from the frame. This does not affect thesterility or the clean room state of the rest of the modular biologicalfoundry system, because the door (i.e. the sealing system) guaranteesthe physical separation between the module that is removed and the restof the modular biological foundry system. This makes this parallelstructure fault tolerant. Adding more modules not only increasesefficiency, but also decreases the risk of overall failure of themodular biological foundry system (by increasing redundancy).

Once new modules that have a better performance for a given task becomeavailable, it is possible to add them to the modular biological foundrysystem without redesigning, modifying or affecting the rest of thesystem. For example, it is possible to add modules that are faster atperforming a given task, if R&D activities develop such modules, or ifbetter machines are commercially available. Likewise, it is possible toadd to the modular biological foundry systems modules that are morereliable than the previous ones. These new modules can simply be addedto the frame of the modular biological foundry system, without costlyand time-consuming redesign processes.

It is also possible to add modules that are different than thepreviously installed modules. For example, it might be necessary toexpand the manufacturing process by adding modules that incorporate newtechnologies. In the cell manufacturing space, and example of an upgradeof this kind might be represented by adding and electroporationmodule—replacing (or paired with) a previously used viral transductionmodule for the genetic engineering step of the cell manufacturingprocess. This extreme flexibility means that the modular biologicalfoundry system is process-agnostic. By accepting new modules—which mightbe completely different from the previous ones—the modular biologicalfoundry system can quickly adapt to new processes and start producingnew kinds of therapies. FIG. 9 shows an extreme example of thisflexibility, in which the same modular biological foundry systemarchitecture can be used to manufacture two completely different typesof pharmaceutical products (cell therapies and personalized capsules, inthis example). The two processes, which have no manufacturing steps incommon, can be implemented simply by mounting on the frame of themodular biological foundry system the right set of manufacturingmodules. The manufacturing modules are completely different for the twomodular biological foundry systems shown in the example in FIG. 9, butthe overall modular structure of the modular biological foundry systemis the same.

This also means that, while a therapy is under development, the modularbiological foundry system has the flexibility to upgrade and improve theprocess by modifying its steps and their sequence. Modifications andupgrades like these do not require engineering or retooling costs(beyond building the modules that will be added). The system that isalready in place is not affected by the additions. This flexibility isextremely important in the cell manufacturing space, because thesemanufacturing processes frequently evolve together with the clinicaldevelopment of the therapies. It is therefore advantageous not to tiedown the design of the process to the original choice of modules, whichmight turn out to be sub-optimal as the drug advances in its clinicalpath. The flexibility of the modular biological foundry system overcomesthis limitation of state-of-the-art manufacturing systems.

Within the modular biological foundry system, the order of themanufacturing operations to be performed on a given batch can be changedsimply by instructing the robotic material transfer system to modify thesequence of modules by which a batch will be processed. This makesiterating on the process sequence and parameters very simple, both onthe hardware side (i.e. the machines used to perform the process can bechanged) and on the software side (i.e. the instructions used to performthe process can be changed, and their order can be changed).

The modular structure of the modular biological foundry system alsomakes it very easy to achieve industrial scale (i.e. high, efficientthroughput) once the first modular biological foundry system has beenoptimized. In this case, scaling is as easy as building new modularbiological foundry systems, each identical to the first one. Thestructure of the modular biological foundry system is extremely modular,so it is very easy to build and assemble additional clusters, and eachone will have the same performance of the first one, because allprocesses are completely automated. In other words, the fully automated(no labor-associated variability) and modular (easy-to-assemble,easy-to-deploy systems) nature of the modular biological foundry systemmakes it ideal to rapidly increase the production capacity for celltherapy products.

Additionally, since the modules and the robots are tightly packed insidemodular biological foundry system, this manufacturing architecturerequires much less space than a traditional labor-based approach. Withina modular biological foundry system there are only machines (no need toleave space for humans), and the modules can be stacked in vertical rows(further increasing machine density and production floor utilization).This results into a very high space efficiency when manufacturing celltherapies with the modular biological foundry system. A single modularbiological foundry system occupies about 200 sq. ft., which is much lessthan a traditional labor-based cell manufacturing suite (which occupiesabout 1,000 sq. ft.). Moreover, the modular biological foundry systemcan realize N product simultaneously (where N is the number of units ofthe slowest module inside modular biological foundry system—usually thebioreactor module). Conversely, a traditional labor-based cellmanufacturing suite can only manufacture one cellular manufacturingtarget at a time (to avoid the risk of mix-ups, human operators can onlymanage one batch).

Therefore, modular biological foundry systems are not only moreefficient than traditional labor-based systems in terms of time and uniteconomics. They are also more efficient in terms of space. This is veryimportant, because cell manufacturing processes require advanced cleanrooms, which are very expensive (per square foot). This makes achievingindustrial scale with modular biological foundry systems economicallymore convenient also from the point of view of the capital investmentsnecessary to build cell manufacturing facilities.

Yet another aspect of the present disclosure is directed to providing adocking device (e.g., docking device instrument) for receiving aportable cell therapy cartridge. The docking device includes a baseincluding a substantially planar upper surface. The substantially planarupper surface is configured to support a lower surface of a portablecell therapy cartridge. The docking device further includes an array ofa plurality of engagement members. Each engagement member protrudesupwardly from the substantially planar upper surface of the base.Moreover, each engagement member in the array of the plurality ofengagement members includes a fixed body protruding upwardly from thesubstantially planar upper surface of the base. Additionally, eachengagement member in the array of the plurality of engagement membersincludes a spring integrally formed with the fixed body and extendinginwardly from an upper end portion of the fixed body oblique to both thesubstantially planar upper surface of the base and the fixed body. Fromthis, a deformable gap is formed interposing between a surface of thefixed body and a lower end surface of the spring. Accordingly, an upperend surface of the spring is configured to engage a surface of theportable cell therapy.

Referring to FIG. 7 through FIG. 10, a docking device instrument 300-1is provided. Each docking device utilizes passive compliance andself-aligning features of engagement members 750. In some embodiments,since a manufacture of cellular engineering targets is automated (e.g.,without human interference) from an initial input to a final output, itis important to keep a portion of the cellular engineering target, whichare inside of rigid cartridges, or other containers, in place before theportion of the cellular engineering target is picked up by the roboticmaterial transfer system, and, similarly, after the portion of thecellular engineering target is disposed (e.g., retrieved from interiorof first module 208-1 and placed inside interior of second module208-2). For instance, in some embodiments, the portion of the cellularengineering targets must be kept substantially stationary within amodule, while an instrument accommodated by the module performs variousmanufacturing tasks to produce the cellular engineering targets.Accordingly, precise and repeatable positioning of instruments andregents within the modular biological foundry system 300 is achieved byusing a docking device 300 that incorporates passive elastic and dampingfeatures via engagement members 750.

The docking device instrument utilized by a modular biological foundrysystem combines a positional accuracy of afforded pins with a toleranceto dimensional errors that is yielded by chamfers. This is achieved withelastic springs (e.g., spring 754 of FIG. 10) that deform when a rigidcartridge is inserted, keeping the rigid cartridge in place and aligninga position of the rigid cartridge based on corresponding positions of anarray of the engagement members, such as automatically and passivelycentering the rigid cartridge on a substantially planar upper surface ofa base of the docking device. Since these engagement members elastic andpassive, they are extremely robust and do not require specialmaintenance to function properly. Moreover, since the engagement membersare designed to operate in an elastic region of a material of theengagement members, the engagement members tolerate a very high numberof cycles of engaging with the rigid cartridge.

By using an array of engagement members 750 all around the cartridge,the position of the rigid cartridge is self-aligned even if anarticulated robot 202 deposits the rigid cartridge on the docking deviceoff-center. Accordingly, when there is a positioning error in theplacement of the rigid cartridge, springs 754 of engagement members 750on one side of the docking device would be more compressed than theother engagement members n an opposing side of the docking device. Fromthis, these compressed engagement members would push the rigid cartridgewith more force, moving the rigid cartridge towards a center of thedocking device, until the force exerted by the spring of each engagementmember in the array of engagement members is balanced. This passiveload-balancing process ensures that the docking device is self-aligning,in that the springs of the engagement members always pushes aninstrument deposited into the docking device towards a center of thedocking device, such as a central axis or a center of the array ofengagement members. As a result, the docking device autonomously (andpassively) eliminates any positioning error that might be resulting fromthe motion of the articulated handling robot and/or transport path ofthe modular biological foundry system.

Another advantage of the docking device of the present disclosure isthat the spring of engagement members push against an instrumentreceived by the docking device. This way, the springs of engagementmembers also exert forces along directions that are perpendicular togravity. Therefore, the instrument received by the docking device issecured in place until the instrument is taken by the articulatedhandling robot. The additional forces exerted by engagement members ofthe docking device on the docked part make the docking device robust tovibrational forces and to external forces that are not in the directionof gravity. Moreover, since the docking device provides passiveengagement members that do not require any external power (e.g.,mechanical power and/or electrical power), nor a active control systems,the docking device of the present disclosure is fault-tolerant and keepsthe instrument received by the docking device in place even in case of apower outage at a modular biological foundry system.

In some embodiments, engagement members 750 are disposed along aperimeter (e.g., contour) of an upper end portion and/or side portion ofthe docking device. In some embodiments, each engagement member of thedocking device has a uniform shape, such that the engagement members arefaster and cheaper to manufacture. Additionally, in some embodiments,each engagement member is removably coupled to the base of the dockingdevice, which allows for modularity of the engagement member makes itpossible to include any number of engagement members as needed to adocking device. For instance, in some embodiments, the array ofengagement members includes at least 3 engagement members, at least 4engagement members, at least 5 engagement members, at least 6 engagementmembers, at least 7 engagement members, at least 8 engagement members,at least 9 engagement members, at least 10 engagement members, at least11 engagement members, at least 12 engagement members, at least 13engagement members, at least 14 engagement members, at least 15engagement members. However, the present disclosure is not limitedthereto. Additional engagement members 750 will result into a strongergripping force exerted on the instrument received by the docking device.Moreover, using a plurality of engagement members allows the dockingdevice to adapt to any shape of instrument. For instance, in someembodiments, the array of engagement members is configured such that thedocketing device is suitable for receiving an instrument that has aprismatic shape, such as an instrument including a substantially planarbase of any shape extruded vertically. However, the present disclosureis not limited thereto. For instance, in some embodiments, the array ofengagement members allows for receiving an instrument having a complexshape, which is completely secured in the docking device by the springsof the engagement members. Accordingly, the docking device can securelyhold instruments with very complex base shapes. In some embodiments,these base shapes of the instruments received by the docking device caneven include cavities (e.g., voids), convex profiles (e.g., a lowersurface of the instrument has a convex curvature), irregularities, oneor more curvilinear edges, or a combination thereof.

Yet another aspect of the present disclosure is directed to providing arigid cartridge. The rigid cartridge includes a plurality of rigid wallsdefining a fixed interior. The plurality of rigid walls includes aplurality of rigid side walls. At least two adjacent edges in aplurality of edges formed by the plurality rigid side walls includes aradius of curvature greater than zero. A substantially planar upperrigid wall connected to an upper edge portion of each rigid side wall inthe plurality of rigid side walls. The substantially planar upper rigidwall includes a plurality of apertures. Each respective aperture in theplurality of apertures is configured to receive and fixedly engage acorresponding connector which is a member of a group consisting of atleast three connectors. Moreover, each respective connector in the atleast three connectors provides communication with a corresponding portin a plurality of ports of the soft body bioreactor. Each respectiveaperture in a first subset of apertures in the plurality of apertures isencompassed by a corresponding annular hood protruding upwardly from thesubstantially planar upper rigid wall by a first height greater than orequal to a second height of the corresponding connector of the at leastthree connectors and integrally formed with the substantially planarupper rigid wall. Additionally, the plurality of apertures includes thefirst subset of apertures. Each respective aperture in the first subsetof apertures is configured to receive and fixedly engage a fluidicconnector of the at least three connectors configured to provide fluidiccommunication with at least a first port in the plurality of ports ofthe soft body bioreactor, and a second subset of apertures. Furthermore,each respective aperture in the second subset of apertures is configuredto receive and fixedly engage an electrical connector of the at leastthree connectors configured to provide electrical communication with atleast a second port in the plurality of ports of the soft bodybioreactor. The rigid cartridge further includes a seamless lip definedby a lower edge portion of each rigid side wall in the plurality ofrigid side walls. Each interior surface in a pair of opposing interiorsurfaces of two rigid side walls in the plurality of rigid side wallsincludes a respective first mating mechanism configured to engage acorresponding second mating mechanism of the soft body bioreactor, suchthat a pair of opposing end portions of the soft body bioreactor isremovably coupled with the rigid cartridge.

Referring to FIG. 12 through FIG. 24, a variety of rigid cartridge(e.g., instrument 300-1 of FIG. 11 are provided, which facilitatessampling of a cellular engineering targeted accommodated by a rigidcartridge and/or a soft body bioreactor, which is performedautomatically by articulated handing robot 204 within the modularbiological foundry system. Accordingly, the rigid cartridges of thepresent disclosure provides repeatability of automatic systems thatensures consistency of manufacture of a cellular engineering targetsince a sample is taken in the same way, such that multiple samples canbe effectively compared, and the variations reflect variations in thecellular engineering target (and not variations in the operators'sampling procedures). This makes it easier and safer to ensure andcontrol the quality of the cellular engineering target.

Specifically, rigid cartridge 1100 includes rigid walls 1110 that definea fixed interior. In some embodiments, fixed interior of rigid walls1110 is utilized to store a portion of a cellular engineering target 2manufactured by biological foundry system (e.g., rigid cartridge of FIG.18C). In alternative embodiments, the fixed interior of the rigid wallsaccommodates a soft body bioreactor that accommodates the cellularengineering target, such as soft body bioreactor 1220 of FIG. 12. Inthis way, the rigid side walls protect the fixed interior of the rigidcartridge (e.g., from contamination and/or external forces, such as anarticulated handling robot). Edges formed by the rigid side wallsincludes a radius of curvature greater than zero, which provided roundedor chamfered edges, allowing for an articulated handing robot to graspthe exterior of the rigid walls of the rigid cartridge device when thearticulated handing robot is not accurate and/or precise in locating therigid cartridge device.

Furthermore, in some embodiments, the rigid cartridge 1100 includessubstantially planar upper rigid wall 1110-2 that is connected to anupper edge portion of each rigid side wall. The substantially planarupper rigid wall includes apertures (e.g., apertures 1180 of FIG. 14).Each respective aperture 1180 is configured to receive and fixedlyengage a corresponding connector (e.g., connectors 1190 of FIG. 15),which allows for communication with the interior of the rigid cartridge.For instance, each respective connector provides communication with acorresponding port in a plurality of ports of the soft body bioreactor.However, the present disclosure is not limited thereto.

For instance, referring briefly to FIGS. 18A and 18B, a variety of rigidcartridges 1100 of a modular biological foundry system are provided.Each rigid cartridge 1100 accommodates a portion of an engineeringtarget (e.g., a cell therapy cellular manufacturing target in liquidform) that must be sampled and/or transferred between rigid cartridges.Accordingly, each rigid cartridge includes rigid walls 1110 that definea fixed interior for accommodating a portion of a cellular engineeringtarget.

In some embodiments, rigid cartridge 1100 includes aperture 1180 thatengages connector 1190 that forms a sampling port. The sampling portconnector 1190 allows for an external sampling system of biologicalfoundry system access to the cellular engineering target accommodated bythe rigid cartridge. In some embodiments, the sampling port connector1190 is separated from the external environment by a gate mechanism(e.g., gate mechanisms 1170 of FIG. 18C). In some embodiments, gatemechanism 1170 provides a sterile sealing mechanism, such as a cap.Accordingly, in accordance with a determination of an instruction (e.g.,communicate via communications network 106 from computer system 100 ofFIG. 1) executing a sampling procedure, articulated handling robot 202opens the gate mechanism 1170, accesses the sampling port connector1190, and removes a sample of the cellular engineering targetaccommodated inside the rigid cartridge. However, the present disclosureis not limited thereto. For instance, in some embodiments, the sampleremoval operation is performed with a sampling container (e.g. a bin, acontainer, a spoon, etc.), with a vacuum-based mechanism (e.g. asyringe, a pipette, etc.) or with another mechanism that allows forremoval of a part of a cellular engineering target accommodated insidethe rigid cartridge. After the articulated handling robot has retrieveda sample of the cellular engineering target, the articulated handlingrobot deposits the sample inside of a sampling container (e.g. a vial, aflask, a petri dish, etc.), such as a second rigid cartridge configuredas a sampling container. The sampling container rigid cartridge includesa similar sampling port connector and a gate mechanism. Accordingly, insome embodiments, after the sample has been deposited into the samplingcontainer rigid cartridge, the dealing system is activated (or put inplace), and the sampling container is ready to be delivered to thequality control operators through the airlock system of the modularbiological foundry system.

Accordingly, articulated handling robot 202 is able to autonomouslyexecute one or more operations in coordination with rigid cartridge 110including, opening gate mechanism 1170, accessing connectors 1190including sampling port connectors, removing a sample of a cellularengineering target from an interior of a first rigid cartridge,depositing the sample into an interior of a second rigid cartridge,disconnecting from the connectors 1190, and closing the gate mechanism1190. However, one of skill in the art will appreciate that the presentdisclosure is not limited thereto. In some embodiments, one or more(e.g., all) of these operations is executed automatically (e.g., viacomputer system 100 in communication with articulated handling robot 202and transport path 202), and in a sterile manner, within the clean-roomenvironment of the modular biological foundry system.

In some embodiments, a rigid cartridge 1100 includes a consumablemechanism (e.g., non-reusable mechanism, such as a perforated seal),ingredient, or reagent that is used during a sampling process.Accordingly, in some embodiments, the consumable mechanism of the rigidcartridge is then autonomously disposed of by the robotic system of themodular biological foundry system. The disposal of consumable is alsoexecuted in an automatic and sterile manner. For example, consumablescan be deposited by the robotic systems into a waste cartridge. Once thewaste rigid cartridge is full, it is can be safely removed by amanufacturing operator from the outside of the modular biologicalfoundry system. Both removing full waste cartridges and replacing themwith empty waste cartridges are operations that are executed withoutaffecting the clean-room, sterile conditions inside the modularbiological foundry system. The waste cartridge can be equipped with itsown sealing system, in order to isolate the waste material from the restof the modular biological foundry system.

In some embodiments, the rigid walls of the rigid cartridge includeincludes polylactic acid (PLA), acrylonitrile butadiene styrene (ABS),polyethylene terephthalate (PET), PET glycol-modified (PETG),polyethylene cotrimethylene terephthalate (PETT), thermoplasticelastomer (TPE), thermoplastic polyurethane (TPU), thermoplasticcopolyester (TPC), nylon, polycarbonate (PC), brass, copper, bronze,aluminum, or iron.

The automatic sampling approach also enables a user of computer system100 to schedule a collection of samples of cellular engineering targetsfrom multiple rigid cartridges 1100. In some embodiments, the collectionof samples is scheduled remotely from the modular biological foundrysystem (e.g., through the communications network 106 that oversees amanufacture of cellular engineering targets at the modular biologicalfoundry system). In such embodiments, the robotic material transfersystem collects the samples from a subset of cellular engineeringtargets defined by the computer system manufacturing at the modularbiological foundry system and place each respective sample into aseparate and sealed rigid cartridge. The rigid cartridges including thesamples are then placed into a mechanism, such as a track, a tray, apallet, or the like that is stored inside the modular biological foundrysystem (e.g., portion of frame 206), until all the samples requested bythe computer system have been collected by the robotic material transfersystem. From this, the robotic material transfer system moves the rigidcartridges including the sample rack into the airlock system (e.g.,first and/or second sealing mechanisms 214, 224), where a user at themodular biological foundry system can retrieve the collection of samples(e.g., on the rack) and then bring the collection of samples to ananalytical lab where quality control operations are performed on thecollection of samples, and similar operations. This way, a user of thecomputer system 100 can schedule a collection of samples and request ananalyze of the collection of samples remotely from the modularbiological foundry system. Moreover, in such embodiments, a user entersthe modular biological foundry system once, in order to retrieve thecollection of samples stored by the sealed rigid cartridges that havebeen prepared by the robotic material transfer system. This approach tothe collection of samples via rigid cartridges 1100 and the roboticmaterial transfer system minimizes a risk for contamination of cellularengineering targets manufactured at the modular biological foundrysystem and allows users to minimize time spent inside the modularbiological foundry system.

Referring to FIGS. 18B and 18C, various embodiments configurations forgate mechanism 1170 of rigid cartridge 1100 are provided, such as tofacilitate a sampling port to access a cellular engineering targetaccommodated within an interior of the rigid cartridge. In someembodiments, the gate mechanism includes a thread, such as a thread onan interior surface of the gate mechanism (e.g., thread 1170-1) or anexternal thread, which allows for the gate mechanism to form a sealsealed with a threaded cap (e.g, gate mechanism 1170-2 of FIG. 18B). Insuch embodiments, the robotic material transfer system can grasp andremovably couple the gate mechanism (e.g, screws/unscrews the cap1170-2) when obtaining a sample of a cellular engineering targetaccommodated by an interior of the rigid cartridge. Referring to FIG.18C, in some embodiments, the gate mechanism includes a sliding gatemechanism, such as a thread less sliding gate. Accordingly, the gamemechanism includes at least an open position, which permits access tothe interior of the rigid cartridge, and a closed position, whichprevents access to the interior of the rigid cartridge. In someembodiments, when the gate mechanism is in the closed position, asealing mechanism (e.g., sealing mechanism 218) ensures that a regionaround the gate mechanism remains sterile and sealed from an externalenvironment when collecting a sample of a cellular engineering targetfrom the interior of the rigid cartridge mechanism. In some embodiments,the sliding gate mechanism includes a spring that engages at a first endportion of the gate mechanism, which forces the gate mechanism towards aclosed position and/or remain stationary. Accordingly, in suchembodiments, if the robotic material transfer system needs to open thesealing mechanism, the robotic material transfer system can push or pullon a protruding feature located (e.g., handle 214 of FIG. 6) on the gatemechanism. A force exerted by the robotic material transfer system onthe protruding feature of the gate mechanism will open the sealingmechanism, providing the robotic material transfer system with access tothe interior of the rigid cartridge, and thus the cellular engineeringtarget. Accordingly, in some embodiments, once an operation forcollection of the sample of the cellular engineering target is deemedcomplete, the robotic material transfer system retracts (e.g., from theinterior of the rigid cartridge and/or from applying the force on theprotruding feature. In some embodiments, such action by the roboticmaterial transfer system causes the spring to push the gate mechanismback in the closed configuration. In some embodiments, the gatemechanism includes a hinged door. In some embodiments, the gatemechanisms includes one or more rotating disks, such as a rotating diskwith corresponding aperture. In some embodiments, the gate mechanismincludes one or more shutter mechanisms. However, the present disclosureis not limited thereto.

Accordingly, the gate mechanism of each rigid cartridge 1100 allows forreusability, in that, the gate mechanism can be used multiple times bythe robotic material transfer system to sample the cellular engineeringtarget accommodated by the interior of the rigid cartridge. Thismulti-use usability of the gate mechanism can guarantee sterility andprotection of the cellular engineering target. Moreover, this gatemechanism is preferable to single-use solutions, because the gatemechanism occupies a smaller volume than other single use mechanisms.Furthermore, the gate mechanism of the rigid cartridge does not limit anumber of samples of the cellular engineering target that can beextracted from the rigid cartridge.

FIGS. 19A, 19B, and 19C shows various possible configurations for achannel 1900 that extends from an aperture (e.g., aperture 1180 of FIG.14) of rigid cartridge 1100. which allows for communication with aninterior of the rigid cartridge. In some embodiments, the channel is atube that connects the gate mechanism with the cellular engineeringtarget accommodated within the interior of the rigid cartridge that isto be sampled. However, the present disclosure is not limited thereto.In some embodiments, the channel includes a rigid channel. In someembodiments, the channel includes a straight channel. For instance, insome embodiments, the channel is rigid and straight, or substantiallystraight. In some embodiments, the channel is non-rigid (e.g.,flexible). In some embodiments, the channel is in non-straightconfiguration, such that a shape of the channel includes be curvilinearshapes, shapes with right angles, or a combination thereof. Accordingly,the channel includes a length that is sufficient to reach a level of aliquid cellular engineering target even when the level is low, or whenthe cellular engineering target is in a corner of the cartridge. Assuch, the shape of the channel can be optimized, so that the roboticmaterial transfer system conducting collecting a sample of the cellularengineering target from the rigid cartridge in an effective and timeefficient manner.

In some embodiments, the robotic material transfer system includes apressure-differential mechanism, such as a syringe and/or a pump, tocollect the sample of the cellular engineering target via suction, whichcauses the channel to fill with the fluid sample of the cellularengineering target. At the end of this sampling procedure conducted bythe robotic material transfer system, having the channel be empty fromresidual fluid is necessary. This emptying of the channel can beachieved by tilting the rigid cartridge that includes the cellularengineering target, such as via articulated handling robot 202 of therobotic material transfer system and/or an actuated base platform offrame 206. In such embodiments, when the rigid cartridge is inclined,gravity pushes the fluid cellular engineering target towards a lower endportion of the rigid cartridge. Accordingly, by controlling theinclination of the rigid cartridge, a designer of the systems andmethods of the present disclosure can ensure that there is no fluid atthe bottom of the channel. This way, the robotic material transfersystem can retrieve the fluid cellular engineering target that remainedin the channel. In some embodiments, as the channel is emptied of fluid,the channel is filled with air as there is no fluid anymore at a base ofthe channel (e.g., open end portion of the channel in the interior ofthe rigid cartridge. Tilting or inclining the cartridge that includesthe cellular engineering target is especially easy if this tiltingprocess makes use of a rocking bioreactor instrument 300 (e.g.,instrument 300-2 of FIG. 12), that includes a substantially planar upperplatform which tilts about an axis.

In some embodiments, in order to retrieve the sample, once a connectionto aperture 1180 that includes channel 119 has been established by therobotic material transfer system, the robotic system can utilize asingle-use pressure differential mechanism, such as a syringe or apipette. These consumable, one-use single-use pressure differentialmechanisms can be stored within (e.g., accommodated within an opening ona surface of the rigid cartridge) the modular biological foundry systemin one or more rigid cartridges that are inserted in pre-determinedslots by manufacturing operators (who are located outside of the modularbiological foundry system). Accordingly, in some embodiments, the rigidcartridge is inserted in and/or removed from via second sealingmechanism 224 of module 208 of frame 206 of the modular biologicalfoundry system without compromising or affecting the clean-room, sterileenvironment inside of the frame. Furthermore, in some embodiments, thesingle-use pressure differential mechanism is sealable, it can be usedalso as the sampling includes. For example, in some embodiments,threaded syringes can be “locked” by screwing a cap onto their threadedopening once the sampling procedure is completed. In such embodiments,after the cap is mounted on the syringe, the assembly (syringe+cap)constitutes a sealed container which can then be delivered a userthrough a sealing mechanism of the modular biological foundry system.

In some embodiments, the pressure differential mechanism (e.g. a syringeor a pipette) is operated by the robotic material transfer system, suchthat the robotic material transfer system includes an articulatedhandling robot 202 and/or flexure gripping device that allows flowcontrolled gripping of the pressure differential mechanism. In this way,the robotic material transfer system must be able to securely grasp andhold the pressure differential mechanism. At the same time, the grippermust also be able to actuate the pressure differential mechanism (e.g.actuate a piston of a syringe). For this reason, in some embodiments,the robotic material transfer system includes a composite gripper devicethat combines a grasping function with a pressure (e.g., suction)control function. This composite gripper device includes at least twoseparate actuation mechanisms to control the at least two separatefunctions, such a first actuation mechanism to control a graspingfunction and second actuation mechanism to control a suction function.

In some embodiments, the modular biological foundry includes a toolchange system what allows the robotic material transfer system toutilize different mechanisms required to interact with one or moremodules 208, one or more instruments 300, one or more rigid cartridges1100, or a combination thereof. For instance, consider if a firstgripper device is substantially different from a second gripper devicethat the robotic material transfer system needs to perform other tasks,it will be necessary to include the tool changing system within themodular biological foundry system. In this way, the robotic materialtransfer system can then use the tool changing system to swap differentgripping devices to change the end effectors of the robotic materialtransfer system. Accordingly, the gripper device that is appropriate fora particular task (e.g., moving a rigid cartridge or sampling a cellularengineering target from the rigid cartridge) is automatically mounted onthe robotic arm before the particular task is performed.

As described supra, the modular biological foundry system includesmodules 208 that are accommodated by frame 206. In some embodiments,each module 208 carries out a single task or a defined set of taskswithin a process to manufacture a cellular manufacturing target based onan instrument 300 accommodated by the module. Accordingly, after themodule has completed the single task or the defined set of tasks, therobotic material transfer system moves the rigid cartridge to a secondmodule 208 that performs a subsequent task or defined set of tasks.

In this way, each module 208 accommodates an instrument 300. In someembodiments, the module also includes one or more support systems thatsupport a task performed by the instrument, such as electronics system(e.g., sensor system), air filter system, and the like. Since differentmodules 208 perform a wide variety of tasks within a manufacturingprocess for producing a cellular engineering target (e.g. a first module208-1 performs centrifugation via a centrifuge instrument 300-1, asecond module 208-2 performs freezing or thawing via a heat exchangeinstrument 300-2, a third module 208-3 includes a bioreactor instrument300-3 to expand a cellular engineering target, etc.). Accordingly, theway in which the robotic material transfer system operates andinterfaces with each module 208 and/or rigid cartridge 1100 is dependenton the specific features of the module and/or the rigid cartridge, orthe instrument accommodated by the module. As such, in some embodiments,in order to allow the robotic material transfer system to operateautonomously with each module in the modular biological foundry system,a corresponding rigid cartridge for each type of module is provided.

As used herein, a “cartridge” is a container where a cellularengineering target and/or one or more reagents that is needed tocomplete a manufacture of the cellular engineering target is storedwithin an interior of the cartridge. The cartridge also allows therobotic material transfer system to easily manipulate the cellularengineering target and its ingredients, picking the cartridge up from afirst module 208 and placing then the cartridge into a second module. Inother words, the cartridges can be easily operated by the roboticmaterial transfer system, which is desired to ensure that the entirecellular engineering target manufacturing process is performedautonomously within the modular biological foundry system. In this way,the cartridge is an interface between the robotic material transfersystem and the instrument that is included within the module. Given thatthe instrument is typically designed to be operated by human users, thecartridge simplifies a task of facilitating operating the instrument viathe robotic material transfer system. Furthermore, by allowing therobotic material transfer system to not only engage the cartridge butalso operate an instrument 300 (instead of a user operating theinstrument), the cartridge is an desired step in guaranteeing thesterility and the repeatability that is provided by the modularbiological foundry system.

In some embodiments, the modular biological foundry system is modularboth from the hardware (e.g., frame 206, modules 208, cartridges 1100,etc.) but also from an ability of the modular biological foundry tointerface and communicate with computer system 100 point of view. Inthis way, the robotic material transfer system connects the variousmodules of the frame within modular biological foundry system by movingone or more cartridges 1100 between different addresses of the moduleswithin the frame. At the same time, in some embodiments, the computersystem 100 manages a flow of materials and manufacture of cellularengineering targets throughout the modular biological foundry system. Insome embodiments, this oversight and management provided by the computersystem is remote. In some embodiments, this oversight and managementrequires constant communication via communications network 106 betweenthe modular biological foundry and the computer system, such thatreal-time data associated with the manufacture of the cellularengineering targets is provided from the biological foundry system tothe computer system. However, the present disclosure is not limitedthereto. In some embodiments, the computer system includes one or moreboard modules that represents each module. Each board includesmodule-specific drivers, which allows the computer system 200 to operatethe instrument 300 that is accommodated in the module. In someembodiments, the board also includes a network interface that allows themodule to communicate with the other modules in the modular biologicalfoundry system, such as via communications network 106.

As described supra, rigid cartridges 1110 of the present disclosureallow different modules 208 to perform different operations with one ormore instruments 300 on the same cellular engineering target, whileprotecting the sterility of the cellular engineering target and,therefore, enabling complete process automation (e.g., manufacture ofthe cellular engineering target without human interference).

Referring briefly to FIG. 20, a view of first rigid cartridge 1100-1that includes cellular manufacturing target 2 is provided. An interiorof the rigid cartridge is defined by rigid side walls 1100 and istypically dedicated to storing the cellular engineering target that isin liquid, or substantially liquid, form, such as a solution includingcells. In some embodiments, the cellular engineering target is in gel,semi-solid, or solid form, and the cartridge is also able to includesthese different forms of the cellular engineering target. However, themost common form of cellular engineering targets is the liquid form—sothe present disclosure will mainly focus on the liquid form. However,one of skill in the art of the present disclosure will appreciate thatthe present disclosure is not limited thereto.

In some embodiments, the cellular engineering target is stored in afixed interior defined by rigid walls 1110 of the cartridge. In someembodiments, the cellular engineering target is accommodated by a softbody bioreactor that is removably coupled with the rigid cartridge(e.g., soft body bioreactor 1200 of FIG. 13). The advantage of a softbody bioreactor is that is does not obstacle the flow of fluids from andinto the rigid cartridge, while, in contrast, a conventional rigidcontainer would need to allow air to come in as fluid exits and air toget out as fluid enters. However, this issue is resolved by includingone or more connectors 1190 that facilitate communication with the softbody bioreactor. For instance, in some embodiments, the connectorincludes a valve that allows for fluid to enter or exit the soft bodybioreactor to enable liquid transfer via the robotic material transfersystem.

An exterior of each rigid cartridge is a defined by rigid walls 1110.The rigidity provided by the rigid walls allows the robotic materialtransfer system to easily engage with the exterior of the rigidcartridge, such as picking the rigid cartridge from a first module 208-1and placing the rigid cartridge in a fourth module 208-4 in accordancewith instructions communicated via computer system 100. Moreover, insome embodiments, the rigid walls of the rigid cartridge also allow therigid cartridge to be placed and secured in a docking device instrument(e.g., docking device 300-1 of FIG. 9), such that engagement members canengage the rigid cartridge without deforming the exterior of the rigidcartridge. Additionally, in some embodiments, the rigid walls of therigid cartridge include the apertures that simplify the engagement(e.g., insertion and/or extraction) of the connector with one or moreports of the soft body bioreactor. In other words, the rigid exterior ofthe rigid cartridge makes provides a simple interface for the roboticmaterial transport system to engage, and therefore move the rigidcartridge about the modular biological foundry system, to interface therigid cartridge with the instruments accommodated by the modules, and tocollect samples of the cellular engineering target from the rigidcartridge.

In some embodiments, the rigid cartridge includes two or more internalsections, which allows for different materials to be stored in eachrespective internal section of the two or more internal sections. Inthis way, each internal section of the rigid cartridge includes one ormore apertures with corresponding connectors 1180, and, optionally,channels 1190, which allows the robotic material transfer system tosample from a respective internal section of the rigid cartridge and toconnect the internal section with other rigid cartridges or systems,such as a reservoir of material. Examples of the connectors include asampling connector, a fluid connector for input of liquid and/or gasinto the rigid cartridge or for output of liquid and/or gas from therigid cartridge. In some embodiments, the connectors enable the roboticmaterial transfer systems to insert one or more sensors into theinterior of the rigid cartridge, such as in contact with the cellularengineering target (e.g., bringing the sensors or probes in contact withthe liquid cell cellular engineering target). Accordingly, theconnectors of the rigid cartridge allow for sterile and automated accessto the interior of the rigid cartridge, such as allowing access to thecellular engineering target.

In some embodiments, in order to safely connect support systems toconnectors 1180, the robotic material transfer system engages a rigidsocket and plug the rigid socket with the connector.

Referring to FIG. 16 and FIG. 20, in some embodiments, connector 1190 issurrounded by a mating mechanism, such as an annular hood (e.g., annularhood 1195), that protrudes from an upper surface of the rigid cartridgein order to guide the motion of the rigid sockets, making theconnection/disconnection of the rigid sockets from the connectors aneasy process, as well as rapid and repeatable for the robotic materialtransfer system. In such embodiments, both the sockets of the roboticmaterial transfer system and the annular hood of the rigid connector arerealized with rigid materials that simplify grasping for the roboticmaterial transfer system. In some embodiments, the annular hoodprotrudes to a greater height than the connector, such that theconnector is protected from horizontal motion of the robotic materialtransfer system. In some embodiments, the annular hood is cylindrical orsubstantially cylindrical. In some embodiments, the annular isconfigured to engage with a socket 2000, which allows for the roboticmaterial transfer system to engage with the annular hood and communicatewith the connector of the rigid cartridge. In some embodiments, theseparts include compliant features (above described docking station),which help with self-alignment and repeatability. By placing a rigidsocket at the end of a flexible channel 1190, it is possible to make itvery simple for a robotic arm to connect that channel to the connectorsof the rigid cartridge. In such embodiments, the robotic materialtransfer system does not have to directly manipulate the flexible tube(manipulating soft objects is problematic for industrial robots).Instead, the robotic material transfer system simply needs to grasp therigid socket and insert the rigid socket in the corresponding connectoror connectors on the rigid upper surface of the rigid cartridge.

Similarly, the connection between the socket of the robotic materialtransfer system and the connector and/or the annular hood of the rigidcartridge can be removed simply by disengaging the parts (e.g.,grasping) and removing the rigid socket. In this way, in suchembodiments, the robotic material transfer system does not interactswith soft parts, but rather the robotic material transfer system simplymanipulates a rigid part (e.g., rigid cartridge, module, etc.), movesthe rigid part, inserts the rigid part, extracts rigid parts, or acombination thereof from a first position in the modular biologicalfoundry system to a second position.

Accordingly, in such embodiments, the sockets 2000 of the robotichandling system have a function in that the sockets allow the roboticmaterial transfer system to repeatably mate flexible parts such as aflexible channel or soft body bag with the connector on the rigidcartridge. Furthermore, the sockets ensure a sterile connection, suchthat the cellular engineering target is protected from the externalenvironment. The sockets also yield a repeatable, multi-use connection.It is possible to connect and disconnect a socket from a connector morethan once. This avoids the problems of single-use systems (i.e. highcosts, significant waste, need for more space, limits to the number ofoperations that can be performed).

In some embodiments, before engaging socket 200 with connectors 1190and/or annular hoods 1195 of rigid cartridge 1100, the robotic materialtransfer system (e.g. articulated handling robot 202) conducts asterilization process on one or more surfaces, such as each surface ofthe socket and/or each surface of the cartridge. In some embodiments,this sterilization process is carried out autonomously by a dedicatedend effector for the robotic material transfer system (or by an add-onmechanism to other end effectors for the robotic material transfersystem). In some embodiments, localized sterilization strategiesutilized by the sterilization process include spraying sterilizingchemicals (e.g. isopropyl alcohol), applying sterilizing gases (e.g.,ozone), using ultraviolet lights (e.g., UV-B and/or UV-C light) or anykind of sterilizing radiation, physically wiping the surface with amaterial, such as a first material imbued with sterilizing chemicals, ora combination thereof.

By sterilizing all the surfaces involved in the socket mating andconnectors access process, the robotic system ensures that all theconnections are sterile. This is essential to guarantee that thecellular engineering target is always included within closed, sterilesystems. This feature meets and exceeds the safety features ofconventional (non-automated) closed systems for the manufacturing ofcell therapies. In fact, traditional closed systems are still operatedby human technicians. Therefore, a big source of contamination is rightin front of the system (the operator). On the contrary, in the modularbiological foundry system structure described here, the closed systems(i.e. systems with tubes, bags, cartridges that accommodates and protectthe cellular engineering target at all times) are operated by anentirely autonomous system within a completely sterile clean-roomenvironment (inside the modular biological foundry system's frame). Thisminimizes the risk of contamination, creating a double barrier—the celltherapy products are protected both by the closed system that surroundsthem, and by the isolated clean-room nature of the modular biologicalfoundry system that includes all products and all machines (but nohumans).

In some embodiments, an additional advantage of using rigid sockets 2000that are placed at the ends of channels 1900 is allowing rigid cartridge1100 to move while a process task is being completed at a respectivemodule 208 via a corresponding instrument 300. For example, in someembodiments, the rigid cartridge includes the cellular engineeringtarget can engage a surface an instrument, such on a rocking bioreactorinstrument 300-2 of FIG. 13 (e.g., within a bioreactor module 208).Accordingly, the instrument includes an upper surface forming a platformthat rocks back and forth, continuously mixing the cellular engineeringtarget accommodated in the rigid cartridge and ensuring that thereagents reach every part of the cellular engineering target whensupplied through the socket via the robotic material transfer system. Insome embodiments, rigid walls 1110 form a seamless lip at a lower endportion of the rigid cartridge, which engages the upper surface of theinstrument to secure (e.g., removably couple) the rigid cartridge andthe instrument. In some embodiments, if these reagents are suppliedthrough the channels that are flexible are connected via the sockets tothe connectors on the upper rigid wall of the rigid cartridge, therocking motion of the rigid cartridge on the upper surface of theinstrument causes no issues. In some embodiments, the same approach ofutilizes the flexible channels and the sockets can be applied toestablish the flow of other liquids and gases as inputs or outputs tothe rigid cartridge, while allowing the rocking motion of the cellularengineering target within the rigid cartridge. This rigid cartridge,which combines flexible elements (e.g., channel 1900, soft bodybioreactor 1200, etc.) and rigid elements (e.g., rigid walls 1110,connectors 1900, sockets 2000, etc.) enables automation of complex tasksat the modular biological foundry system that are usually performed byoperators.

The connectors of the rigid cartridge can be of any of the typespreviously described (see the section about sampling connectors). Theconnectors of the rigid cartridge can also be connected to any of thepreviously described types of channels. The socket-connectorsarchitecture ensures a sterile, repeatable connection that can beestablished multiple times (reusable) by a fully automated system.

The cartridge configuration that has been described so far is ideal foruse in a bioreactor module. Different cartridges can be designed fordifferent purposes, and in order to adapt to different pieces ofmachinery. In this approach, every module within the modular biologicalfoundry system can have its own cartridge, allowing the completeautomation of each step of the manufacturing process. When possible, thesystems and methods of the present disclosure can also design “generalpurpose” cartridges that are able to interface with multiple differentmodules.

An example of a different type of cartridge is represented by the liquidreservoir cartridge (e.g., FIG. 21). This type of cartridge includes oneor more internal sections. Each section includes a particular liquid—itcould be an input material (e.g. media for cell culture) or an outputmaterial (e.g. waste). Each section is connected to one or more flexiblechannels through which liquid can exit the cartridge or enter thecartridge. All the channels connected to the various sections of thecartridge are bundled together in a single, flexible channel. At the endof it, there is a socket which includes a connectors interfacetermination for each fluid channel. The rigid socket that can be graspedby a robotic manipulation system and inserted into a rigid cartridge.This single insertion operation immediately established contact amongmultiple channels (originating from the cartridge) and multipleconnectors on the cartridge.

In some embodiments, the socket can also be equipped with valves thatstop the flow of fluid. The valves can be operated by the roboticmaterial transfer system, by pushing or moving features (e.g. buttons,levers, etc.) on the outside of the socket. This way, the robotic armcan both insert the socket, and allow/block the flow of illiquid byoperating the valves. Moreover, the presence of valves at the end of thechannel (just before it is connected to the connectors on the rigidcartridge) eliminates the risk for liquid spills andcross-contamination. In some embodiments, the channel includes one-wayvalves, in order to ensure that the flow along a channel can only go ina pre-determined direction. And by adding filters, it is possible toprevent unwanted product components or reagents travel along channelswhere their presence must be avoided.

In some embodiments, one or more the flexible channels that originatefrom a liquid reservoir cartridge can be bundled together to form asingle, flexible channel bundle and to avoid getting caught inprotruding mechanical features. This channel bundle must have enoughslack to enable the robotic material transfer system to grasp thesocket, move it and insert it into its destination cartridge. At thesame time, the channel bundles dimensions and its overall length aredesigned to avoid getting caught in other machines as the roboticmaterial transfer system takes care of the connection/disconnectionprocesses. Additionally, the channel bundle must also provide enoughslack to enable the destination cartridge (to which the socket isconnected) to move (e.g. on a rocking bioreactor instrument).

The channels that originate from a liquid reservoir cartridge (e.g.,rigid cartridge 1100-4 of FIG. 21) can be designed so that the channelspass in front of a rigid surface that mimics the outer shape of aperistaltic pump instrument (e.g., instruments 300 of FIG. 21). Thisway, when the liquid reservoir rigid cartridge is placed on theperistaltic pump instrument by the robotic material transfer system, thechannel will be automatically inserted in a portion of the peristalticpump instrument. In some embodiments, a mating surface of the rigidcartridge, closely following an outer surface of the peristaltic pumpinstrument, will then keep the channel in place. In some embodiments, arigid cartridge with multiple sections (i.e. accommodating multipleseparate liquids) includes multiple channels. In this case, all thechannels can be inserted in multiple peristaltic pump instruments at thesame time. For this to occur, the rigid cartridge is placed by therobotic material transfer system on a set of multiple differentperistaltic pumps instrument (e.g., one for each channel in which adesigner of systems and methods of the present disclosure wants tocontrol liquid flow through). And each channel must be kept in place bya mechanical feature on the outer surface of the rigid cartridge thatmimics the outer shape of the peristaltic pump (e.g., FIG. 21). Thisway, the flow of each liquid in each channel can be controlledindependently by separate peristaltic pump instruments.

Accordingly, this rigid cartridge isolates the flow of each liquid in aseparate closed circuit, protected from the external environment. Inother words, all fluids traveling from or to the liquid reservoir rigidcartridge are always accommodated within a closed, sterile set ofchannels. This approach meets or exceeds all the requirements oftraditional closed systems for the manufacturing of cell therapies, inthat this approach is completely automated—achieving superior safety(requires no human users; no sources of contamination) and superiorefficiency.

In some embodiments, different partitions inside of the rigid cartridge(each including a different fluid) are defined by rigid walls 110. Insome embodiments, the interior partitions include a mechanism thatallows fluids to flow in and from the partitions without resistance. Forexample, in some embodiments, each interior partition of a rigidcartridge includes a valve and/or a filter mechanism. In particular, thevalve lets air in as the stored liquid exits the reservoir. Likewise,the valve lets air out as new fluid enters the reservoir. The presenceof the filter mechanism ensures that the air going through the valvedoes not contaminate the product, or the external environment. The sameeffect can be obtained by adding a flexible membrane (diaphragm) to acartridge section, or by implementing the reservoir through a soft bodybioreactor that is accommodated by the rigid cartridge (in this case, aseparate soft body bioreactor is needed for each fluid reservoir insidethe cartridge). The flexible membrane and the soft body bioreactor canexpand and contract, and do not oppose fluid coming in or getting out.

Another example of a special-purpose cartridge is a cartridge that isdesigned to automate the operation of a centrifuge (e.g., FIG. 22). Acentrifuge for cell therapy manufacturing must transfer the cellularengineering target from an input reservoir to a centrifugation vessel,where the different components of the cellular engineering target areseparated by density. Then, a piston pushes the different components ofthe cellular engineering target out of the centrifugation vessel, andinto output reservoirs. The whole process must be executed in a sterile,closed system of channels and reservoirs.

The centrifugation cartridge includes a section to store the inputmaterial (this section accommodates the initial cellular engineeringtarget at the beginning of the centrifugation task). The centrifugationcartridge is also equipped with one or more sections for the variousparts of the output of the centrifugation task (these sections start ourempty and are then progressively filled during the centrifugation task).All the different sections of the cartridge are connected to thecentrifugation vessel through tubing and a multi-directional valve. Thevalve can be controlled from the outside of the cartridge by the roboticmaterial transfer system (by operating a knob or a lever). This way, therobotic material transfer system can select the valve configuration thatconnects the centrifugation vessel with the cartridge section that isneeded at a particular point of the centrifugation task. Note that thewhole cartridge can be placed on one or more peristaltic pumps (like thefluid reservoir cartridge described before). Once the channels lock ontothe peristaltic pumps, they can be used to control the flow rate of theliquids from/to the reservoirs.

To begin the process, the robotic material transfer system turns thevalve so that the input liquid drips (or is moved by a peristaltic pump)into the centrifugation vessel. Once the fluid reaches thecentrifugation vessel, the centrifuge starts rotating it. This separatesthe different components of the cellular engineering target by density.At this point, the piston moves upwards, pushing out of thecentrifugation vessel one fraction of blood at a time (e.g., FIG. 22).Now, the robotic arm changes the position of the valve, so that thefractions of cellular engineering target that have been separated by thecentrifuge can be directed into one or more separate output reservoirs.This process is repeated until all the useful fractions of the cellularengineering target have been pushed into the output reservoirs. In thiscase too, the presence of additional peristaltic pumps can help controlthe flow rate of the output liquids.

The reservoirs of liquid that are present into the centrifugationcartridge can be implemented either as soft bags accommodated a hardcartridge shell, or as hard containers that are equipped with valves andfilters that let air in or out in order not to obstruct the motion ofthe fluids. It is also possible to substitute the valves and filterswith soft membranes (diaphragms).

In some embodiments, multiple rigid cartridges 1100 can be positioned inthe same module by the robotic material transfer system (e.g.articulated handling robot 202). Accordingly, all the rigid cartridgesthat have been placed inside of the module can be liked together by arobotic material transfer system, forming a single closed system, suchas by linking one or more connectors 1190 from each rigid cartridge inthe module 208. This combined, modular closed system unites multiplerigid cartridges and systems by using the sockets that are grasped bythe robotic material transfer system, moved to a target location, andinserted in or extracted from a target rigid cartridge.

For example, referring briefly to FIG. 23, in some embodiments, a rigidcartridge is disposed on (e.g., lip of rigid cartridges engages) anupper surface of a rocking bioreactor instrument, such that a lip of therigid cartridge engaging an upper surface of the instrument. Theconnectors of the rigid cartridge allow the robotic material transfersystem to sample the cellular engineering target whenever requested (orpre-scheduled) by a user at computer system 100. Accordingly, therobotic material transfer system engages the rigid cartridge with asecond rigid cartridge (e.g., a liquid reservoirs material rigidcartridge), using the sockets. As such, these additional inputs andoutputs allow the system to add materials to the cellular engineeringtarget or remove materials from interior of the rigid cartridge via theconnectors. The connectors also allow the robotic material transfersensor to place sensors in the interior of the rigid cartridge, ifneeded. Since these connectors are sterile, the closed-system nature ofthe rigid cartridge is always guaranteed.

This modular closed-system architecture allows the combination ofmultiple cartridges and systems to automate every task of themanufacturing process of producing cellular engineering targets, whileensuring that the cellular engineering target is always accommodated insterile channels and interior partitions of a rigid cartridge that arephysically separated from the external environment by rigid walls 11100.

In some embodiments, a channel can bundle multiple smaller channelstogether, to form a coupling of channels. For instance, in someembodiments, each channel includes slack (e.g., excess length) andincludes a flexible material that allows the robotic material transfersystem to move the socket and connect it to one or more connectors in acorresponding rigid cartridge. The rigid socket can also include valves(e.g., the valves can be automated, or they can be operated by therobotic material transfer system with mechanical features that arepresent on an exterior surface of the socket).

In some embodiments, the instruments include peristaltic pumps, thatallow the channel tubing to interface with peristaltic pumpsinstruments, that in turn control the flow rate of the fluids inside thechannels and the interior of the rigid cartridge.

In some embodiments, the rigid cartridges 1100, connectors 1190,channels 1900, and sockets 2000 can be combined and connected by therobotic material transfer system, in order to realize a larger closedmodular biological foundry system that allows a cellular engineeringtarget manufacturing task to be carried out. As such, multiple rigidcartridges and subsystems can be connected using sockets and connectors.In some embodiments, peristaltic pump instruments can engage thechannels regulate flow rate. In some embodiments, the valves in thesockets and in the connectors help the system constrain the direction ofthe flow in the channels. In some embodiments, the rigid cartridges canbe connected with other rigid cartridges (or placed inside of otherrigid cartridges) to achieve more advanced, composite functions.

In some embodiments, by automatically combining the above describedelements, a robotic material transfer system can dynamically create anykind of closed system at a modular biological foundry system and enablea cellular engineering target manufacturing task to be carried out. Thisapproach is very general, and enables the automatic assembly anddisassembly of sterile closed systems at the modular biological foundrysystem that can: store fluids in separate rigid cartridges; transferfluids (with a controlled flow rate) between rigid cartridges; andprocess fluids (e.g., modifying the cellular engineering target as partof the various cell therapy manufacturing tasks).

Referring briefly to FIG. 21, an example of a large modular closedsystem that is automatically assembled by a robotic inside of a module(in the modular biological foundry system). In some embodiments, a firstliquid reservoir rigid cartridge is be connected by a robotic materialtransfer system (e.g. a robotic arm) to a set of peristaltic pumpinstruments, and then plugged by the robotic material transfer systeminto the second rigid cartridge. This allows the modular biologicalfoundry system to supply new reagents to the cellular engineering targetof the second rigid cartridge, and to remove waste liquid.

In some embodiments, a liquid cabinet (e.g., FIG. 23) can be pluggedinto the rigid cartridge (e.g., instead of the liquid reservoir rigidcartridge). The liquid cabinet is much larger than a cartridge and caninclude a much larger quantity of input or output liquids than theliquid reservoir cartridge. The liquid cabinet also includes integratedpumps that control the flow to and from its reservoirs. In someembodiments, this is different from the liquid reservoir cartridge,which is designed to be used for a single cellular engineering target(or for a limited number of cell therapy products), because its size islimited by its volume and by the payload of the robotic materialtransfer system. On the other hand, the liquid cabinet is designed toservice a large number of different cellular engineering targetsproduced at the modular biological foundry system, because its volumeand weight can be much bigger than those of a cartridge, and its pumpingsystems are already integrated in its channels. The liquid cabinet canbe plugged into multiple cartridges by a robotic material transfersystem, one after the other (as they are transported into the module bythe robotic material transfer system). This is also enabled by thesocket system, which is sterile and multiuse. Additionally, the valvesin the ensure that the flow always goes in the right direction.Unidirectional valves can also be added to the socket, in order toprevent fluid backflow in an undesired direction (this reduces the riskof spills and cross-contamination).

In some embodiments, a gas cabinet (e.g., FIG. 23) with gas reservoirscan be plugged by a robotic material transfer system into a large numberof cartridges, one after the other, as the robotic material transfersystem brings them into that module. The gas cabinet includes gasreservoirs for both input gases and output gases. It also includesintegrated compressors, gas mixers, filters, and all the gas processingequipment that is necessary to support the cell manufacturing taskcarried out in that module.

The reservoirs of the liquid and gas cabinets can be replenished (oremptied) from the outside of the modular biological foundry system by auser. In this way, these refill and/or emptying operations do not affectthe clean room conditions and the sterility of the modular biologicalfoundry system.

In some embodiments, completing the manufacturing process for a singlecellular engineering target requires a rigid cartridge that accommodatesthe engineering target to go through multiple modules 208. This meansthat the cellular engineering target can require to be carried inmultiple rigid cartridges, one after the other, according to the rigidcartridge configuration needed by the modules that are used to implementthe manufacturing process. While a single cellular engineering targetmight need multiple cartridge, a single rigid cartridge can only be usedfor a single cellular engineering target. After it has been used for acellular engineering target, a cartridge must be extracted from themodular biological foundry system and either disposed of or cleaned andsterilized before being ready to be used for another cellularengineering target.

In some embodiments, the modular biological foundry system can beequipped with slots where bins of cartridges (e.g., sealed binsincluding empty, clean cartridges) can be inserted. In some embodiments,the modular biological foundry system includes slots for bins where therobotic material transfer system deposits used rigid cartridges, suchthat these used cartridges are then removed with the bin, such as oncethe bin is full.

In some embodiments, adding bins with new cartridges or removing binswith old cartridges can be done by a manufacturing operator from outsidethe modular biological foundry system, without affecting the clean-roomsterile conditions inside the modular biological foundry system.

In some embodiments, the use of multiple rigid cartridges for a singlecellular engineering target determines the need for a module 208 withinthe modular biological foundry system in which a cellular engineeringtarget is transferred from an interior of one cartridge to an interiorof a second cartridge in a sterile and automated manner (e.g., within aclosed system of the modular biological foundry system, and with aprocess that is entirely executed by the robotic material transfersystem), hereinafter known as a “cartridge transfer module.”

In some embodiments, in the cartridge transfer modules, a liquidcellular engineering target can be moved from one cartridge (e.g.,origin cartridge) to another cartridge (e.g., destination cartridge) bysimple automatic liquid transfer, such as connectors 1900 and sockets2000, including, but not limited to, pipettes and syringes, operated bya robotic material transfer system, are able to transfer the liquidcellular engineering target from an output (or sampling) connectors inthe origin cartridge, to an input connectors in the destinationcartridge. In some embodiments, a limit of pipettes and syringes is thatthey can only move a limited amount of liquid at a time. Therefore, whenusing these devices, the liquid transfer process must be repeatedseveral times, until the origin cartridge is empty, and the destinationcartridge is full. Since the cartridge transfer module is completelyautomated, these operations can be executed within the module, withoutrequiring the involvement of the robotic material transfer system (e.g.the articulated handling robot 202).

However, if the volume of liquid to be transferred from the origincartridge to the destination cartridge is significant, it might take toomany iterations to complete the transfer with that have a smallvolumetric capacity (i.e. pipettes or syringes). In this case, thecartridge transfer process can be completed quickly by establishing acontinuous flow of liquid between the two rigid cartridges. When it isnecessary to establish a continuous flow of liquid between two rigidcartridges, another embodiment can be implemented in the cartridgetransfer module (e.g., FIG. 24). In this approach, a robotic materialtransfer system places a transfer channel cartridge (which is designedfor a one-time use) onto a peristaltic pump instrument. The roboticmaterial transfer system achieves this by grasping the rigid pump socketthat holds a flexible channel (e.g., FIG. 24). At the two extremities ofthe flexible channel there are rigid sockets 2000, which engage theconnectors of each rigid cartridge, such as each fluid connector. Therobotic material transfer system (e.g., a robotic arm, or a roboticgantry) then engages a socket of the channel with an output connect(from where liquid will be extracted) of the origin cartridge and asecond socket of the channel with an input socket (from where liquidwill be inserted) of the destination cartridge.

In some embodiments, the electrical connector of the rigid cartridgeincludes a pressure control mechanism (e.g., valve), a pH sensor, adissolved oxygen sensor, a temperature sensor, a flow rate sensor, amass sensor, or a combination thereof.

In some embodiments, the fluidic connector of the rigid cartridgeincludes a pressure control mechanism, an inlet port, a sampling port,an outlet port, or a combination thereof. In some embodiments, thefluidic connector includes a valve configured to control a flow of afluid through a corresponding fluidic port.

In some embodiments, a first internal diameter at an upper end portionof a respective connector is less than a second internal diameter at alower end portion of the respective connector. This allows the roboticmaterial transfer system to engage the connector easily given thedefined curvature.

In some embodiments, at least one rigid wall in the plurality of rigidwalls further includes a gate mechanism configured to provide access tothe fixed internal cavity of the rigid housing. In some embodiments, alength of the gate mechanism is greater than or equal to across-sectional area of the soft body bioreactor 1200, which allows forthe soft body bioreactor to be received by the interior of the rigidcartridge from at least on orientation of the soft body bioreactor.

In some embodiments, each rigid coupling mechanism protrudes from thesubstantially upper planar surface of the at least one rigid wall of therigid housing.

In some embodiments, the rigid cartridge further includes acorresponding cap (e.g., cap 1198) for each respective connector in theplurality of connectors. The corresponding cap encompasses therespective connector, which ensure that the cap is sterile. In someembodiments, the corresponding cap is configured to disengage theconnector when a force is applied in a first direction, such as ahorizontal direction.

In some embodiments, an exterior surface of the corresponding capincludes a third mating mechanism confirmed to engage a fourth matingmechanism of an articulated handling robot. In some embodiments, thefourth mating mechanism includes a groove or a protrusion around acircumference of the corresponding cap. In some embodiments, thecorresponding cap is configured to disengage the respective connector inthe plurality of connectors when subject to a lateral force.

In some embodiments, each rigid coupling mechanism of each respectiveport in the at least one electrical port is a push coupling mechanism.

In some embodiments, the robotic material transfer system then actuatesone or more valves of the socket, so that flow is allowed from theorigin cartridge to the destination cartridge. In some embodiments, thevalve is a one-way valve, in order to regulate the flow according to therequirements of the process. Moreover, in some embodiments, the valveinclude a filter to reduce the risk of contamination. Once thecartridges are connected and set up by the robotic material transfersystem, a continuous and sterile connection is established from theorigin cartridge to the destination cartridge. Accordingly, theperistaltic pump can activated and transfer the liquid from the origincartridge to the destination cartridge. Once the transfer is completed,the robotic material transfer system can close the valves of the socketvalves, disengage the sockets from one or more connectors of the rigidcartridge, disposes of the origin cartridge, disposes of the transferchannel cartridge, or a combination thereof. From this, the roboticmaterial transfer system moves the destination cartridge (including thecellular engineering target) into the next module required by amanufacturing process.

In some embodiments, each cartridge, each module and in general eachmachine in the modular biological foundry system can be equipped with acorresponding barcode that communicate identity information (ID) and/orspatial positioning information (e.g., addresses of FIG. 2A). In someembodiments, multiple cameras or vision systems is disposed onstationary or mobile assemblies (e.g. on the gripper device of therobotic material transfer system, in the modules, on the frame of themodular biological foundry system, etc.). With this video sensors andthe corresponding barcode, each cellular engineering target and eachstep of the process of manufacture of cellular engineering targets areautomatically tracked by the robotic material transfer system and/orcomputer system 100. Therefore, the robotic material transfer systemautomatically maintains a full, real-time audit trail for each cellularengineering target. Each step in the process, one or more numericalvalues (or measurements) associated with the manufacturing step (e.g.,temperature, concentration, elapsed time, velocity, etc.), and/or a timestamp is automatically logged and communicated via communicationsnetwork to the computer system 100.

This level of real-time, automatic tracking enables the roboticmanufacturing system to guarantee that there is never confusion betweenmultiple cellular engineering targets. This is a significant differencewith respect to labor-based systems. Manufacturing processes that relyon human operators can only produce one cellular engineering target at atime in order to avoid mix-ups. The robotic material transfer systemdescribed herein can manufacture multiple cellular engineering targetsin parallel (at the same time), because each of the cellular engineeringtargets is completely tracked by the barcode tags and by the visionsystem of modular biological foundry system. It is also possible toimplement the tracking system using radio-frequency tags, magnetic tagsor other types of tags that do not rely on video data. In any case, itis the presence of tags and of an automatic cellular engineering targettracking system that ensure the absence of mix-ups and enable theparallel manufacturing of different cell therapy products.

The foregoing descriptions of specific exemplary embodiments of thepresent invention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteachings. The exemplary embodiments were chosen and described in orderto explain certain principles of the invention and their practicalapplication, to thereby enable others skilled in the art to make andutilize various exemplary embodiments of the present invention, as wellas various alternatives and modifications thereof. It is intended thatthe scope of the invention be defined by the Claims appended hereto andtheir equivalents.

REFERENCES CITED AND ALTERNATIVE EMBODIMENTS

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The present invention can be implemented as a computer program productthat includes a computer program mechanism embedded in a non-transitorycomputer-readable storage medium. For instance, the computer programproduct could contain instructions for operating the user interfacesdescribed with respect to FIG. 2. These program modules can be stored ona CD-ROM, DVD, magnetic disk storage product, USB key, or any othernon-transitory computer readable data or program storage product.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical applications, to thereby enable others skilled in the artto best utilize the invention and various embodiments with variousmodifications as are suited to the particular use contemplated. Theinvention is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled.

What is claimed is:
 1. A rigid cartridge for housing and removably coupling with a soft body bioreactor, wherein the rigid cartridge comprises: a plurality of rigid walls defining a fixed interior; wherein the plurality of rigid walls comprises: a plurality of rigid side walls, wherein at least two adjacent edges in a plurality of edges formed by the plurality rigid side walls comprises a radius of curvature greater than zero, and a substantially planar upper rigid wall connected to an upper edge portion of each rigid side wall in the plurality of rigid side walls, wherein the substantially planar upper rigid wall comprises: a plurality of apertures, wherein: each respective aperture in the plurality of apertures is configured to receive and fixedly engage a corresponding connector which is a member of a group consisting of at least three connectors, each respective connector in the at least three connectors provides communication with a corresponding port in a plurality of ports of the soft body bioreactor, and each respective aperture in a first subset of apertures in the plurality of apertures is encompassed by a corresponding annular hood protruding upwardly from the substantially planar upper rigid wall by a first height greater than or equal to a second height of the corresponding connector of the at least three connectors and integrally formed with the substantially planar upper rigid wall, and wherein the plurality of apertures comprises: the first subset of apertures, wherein each respective aperture in the first subset of apertures is configured to receive and fixedly engage a fluidic connector of the at least three connectors configured to provide fluidic communication with at least a first port in the plurality of ports of the soft body bioreactor, and a second subset of apertures, wherein each respective aperture in the second subset of apertures is configured to receive and fixedly engage an electrical connector of the at least three connectors configured to provide electrical communication with at least a second port in the plurality of ports of the soft body bioreactor; and a seamless lip defined by a lower edge portion of each rigid side wall in the plurality of rigid side walls, and wherein: each interior surface in a pair of opposing interior surfaces of two rigid side walls in the plurality of rigid side walls comprises a respective first mating mechanism configured to engage a corresponding second mating mechanism of the soft body bioreactor, thereby removably coupling a pair of opposing end portions of the soft body bioreactor with the rigid cartridge.
 2. The rigid cartridge of claim 1, wherein the electrical connector comprises a pressure control mechanism, a pH sensor, a dissolved oxygen sensor, a temperature sensor, a flow rate sensor, a mass sensor, or a combination thereof.
 3. The rigid cartridge of claim 1, wherein the fluidic connector comprises a pressure control mechanism, an inlet port, a sampling port, an outlet port, or a combination thereof.
 4. The rigid cartridge of claim 1, wherein, the fluidic connector comprises a valve configured to control a flow of a fluid through a corresponding fluidic port.
 5. The rigid cartridge of claim 1, wherein a first internal diameter at an upper end portion of a respective connector is less than a second internal diameter at a lower end portion of the respective connector.
 6. The rigid cartridge of claim 1, wherein at least one rigid wall in the plurality of rigid walls further comprises a gate mechanism configured to provide access to the fixed internal cavity of the rigid housing.
 7. The rigid cartridge of claim 6, wherein a length of the gate mechanism is greater than or equal to a cross-sectional area of the soft body bioreactor.
 8. The rigid cartridge of claim 6, wherein the gate mechanism is a hinge gate mechanism or a hinge gate mechanism.
 9. The rigid cartridge of claim 1, wherein each rigid coupling mechanism protrudes from the substantially upper planar surface of the at least one rigid wall of the rigid housing.
 10. The rigid cartridge of claim 9, further comprising a corresponding cap for each respective connector in the plurality of connectors, wherein the corresponding cap encompasses the respective connector.
 11. The rigid cartridge of claim 10, wherein an exterior surface of the corresponding cap comprises a third mating mechanism confirmed to engage a fourth mating mechanism of an articulated handling robot.
 12. The rigid cartridge of claim 10, wherein the corresponding cap is configured to disengage the respective connector in the plurality of connectors when subject to a lateral force.
 13. The rigid cartridge of claim 1, wherein each rigid coupling mechanism of each respective port in the at least one electrical port is a push coupling mechanism. 